Universidad Politécnica De Madrid Osama Galal Sakr

UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

BIOSTIMULATION METHODS ASSOCIATED

WITH EARLY WEANING AND REPRODUCTIVE

RHYTHMS IN PRIMIPAROUS RABBIT DOES

TESIS DOCTORAL

OSAMA GALAL SAKR

Ingeniero Agrónomo

2012

UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

DEPARTAMENTO DE PRODUCCIÓN ANIMAL

BIOSTIMULATION METHODS ASSOCIATED

WITH EARLY WEANING AND REPRODUCTIVE

RHYTHMS IN PRIMIPAROUS RABBIT DOES

OSAMA GALAL ABD EL AZIZ SAKR

Ingeniero Agrónomo

Directores de la Tesis:

Pilar García Rebollar Pedro Luis Lorenzo González

Dra. Veterinaria Dr. Veterinaria

2012

DEDICATION

I dedicate this work to whom my heartfelt thanks to my lovely wife Eman and my sweet children Yahya and

Yara for their patience, help, and for all the support they

lovely offered along the period of my departure, as well as to

my late father, my mother and brothers.

Osama G. SaKR

ACKNOWLEDGEMENT

I wish to express my sincere thanks, deepest gratitude and appreciation to Dr. Pilar G. Rebollar and Dr. Pedro L. González for supervision, continued assistance, and their guidance through the field work and revision of the thesis manuscripts. Thanks to Dr.Rosa Garcia and Dr.Maria Arias, for everything, for encouraging me, for helping me and for having the time to teach me the techniques. To Dr. Pilar Millán, thanks for your help in laboratory analysis. Grateful appreciation is also extended to all staff members of Animal Production Department (UP Madrid) and (AECID ) for their collaboration. Special deep appreciation is given to my late father, my mother, my wife, my children, my brothers, my family and my in-laws. Also I feel deeply grateful to my dear country Egypt and Cairo University, Faculty of Agriculture and the Animal Production Department. Thanks to Dr. Nagwa A. Ahmed, Dr. Taha M. Elbedawy, Dr. Shukry Tantawy and Dr. Ahmed Elfar. Deep thanks are given also to all my friends and colleagues in Spain or Egypt especially for, M. Frikha, Martina Pérez, Lourdes Cámara and Julio Francisco for their help and cooperation.

Many thanks to all

AGRADECIMIENTOS

Esta Tesis Doctoral es fruto de un esfuerzo conjunto de todos vosotros que me habéis ayudado de una u otra forma. Gracias por estar ahí y por dejarme compartir con vosotros momentos que nunca olvidaré.

A mi mujer y mis niños. Por vuestro apoyo incondicional y por el esfuerzo que habéis hecho por darnos siempre lo mejor. Gracias Eman, compartir la vida contigo es un aprendizaje continuo.

Gracias Yahya y Yara, la luz de mis días.

A mi madre y mi padre, gracias por vuestros consejos, por estar ahí apoyándome siempre con todas mis decisiones, por vuestra infinita paciencia conmigo a lo largo de este periodo tan duro...y durante toda mi vida... porque si soy lo que soy, es gracias a vosotros.

A mis tutores Pilar García Rebollar y Pedro Lorenzo González quiero agradecerles su confianza y apoyo, así como permitirme aprender y formar parte de su grupo de trabajo en todas las etapas durante mis estudios de Doctorado en España.

A Rosa García, gracias por todo… por dedicarme su tiempo en enseñarme las técnicas, por tu ayuda, A María Arias, gracias por sus ánimos, y su optimismo.

A Pilar Millán, gracias por su ayuda en los análisis de laboratorio.

En la nave, en el laboratorio y en las oficinas siempre tuve unos buenos consejeros. Gracias

Juan Ramon Astillero, Beatriz Velasco, Andrés Caldas y Ángel Revilla.

A la Universidad Politécnica de Madrid por admitir mi participación en el Programa de

Doctorado en el Departamento de Producción Animal de la Escuela Técnica Superior de Ingenieros

Agrónomos, para ampliar mi formación profesional.

A la Universidad del Cairo, Facultad de Agricultura por financiar y darme la oportunidad de realizar mis estudios de Doctorado en España. A los Dres. Nagwa A. Ahmed, Taha M.

Elbedawy, Shukry Tantawy and Ahmed Elfar. A mis colegas y compañeros, los Profesores del

Departamento de Producción Animal.

A la Agencia Español de Cooperación Internacional para el Desarrollo (AECID) por el soporte económico recibido a través de la Beca disfrutada durante estos cuatro años.

Agradezco a Carlos Buxadé, Carlos de Blas, Rosa Carabaño, Nuria Nicodemus, Javier García,

María Jesús Villamide, David Menoyo, Miguel Ángel Toro, Morris Robinson, Paloma García,

Gonzalo G. Mateos, Rosa Lázaro, María Alvir, Javier González, Rafael Alenda, Argimiro Daza,

Ismael Ovejero y Carlos Rodríguez profesores del Departamento de Producción Animal de la

E.T.S.I.A., UPM por las clases, los apoyos, los sabios consejos y los buenos momentos.

A mis amigos, que me habéis acompañado y apoyado en este largo caminar. Nunca olvidaré los buenos momentos vividos, por hacerme sentir su cercanía en los momentos más difíciles y también en los más felices, gracias Mohamed Frikha, Martina Pérez, Lourdes Cámara, Julio Francisco, Nelly

Pereda, Javier López, Rebeca Marcillo, Nourhan El Abed y Rodrigo Abad.

... Finalmente, a todas aquellas personas que han estado cerca en todo este tiempo, y a las que he encontrado al final del camino, por aguantar los muchos altibajos por los que he tenido que pasar sobre todo en la recta final. Gracias cielo.

A quien haya podido olvidar.

A todos los que alguna vez tuvieron una sonrisa A todos, muchas gracias

INDEX

INDEX OF CONTENTENTS

Page

1 ABBREVIATION LIST

RESUMEN 7

SUMMARY 13

CHAPTER 1ː LITERATURE REVIEW AND OBJECTIVES

1. Generalities 21

2. Rabbit reproductive physiology

2.1. Oogenesis and follicular development 22

2.2. Oocyte maturation 26

2.2.1. Nuclear maturation 26

2.2.2. Cytoplasmic maturation 28

2.3. Steroidogenesis in cumulus-oocyte complexes (COC) 33

2.4. Follicular atresia 34

2.5. Oestradiol-17β (E2) and Progesterone (P4) 37

2.6. Ovulation and corpus luteum formation 39

3. Reproductive Management Strategies

3.1. Effect of remating intervals on reproductive physiology of lactating

rabbits 41

3.2. Effect of estrus synchronization on reproductive physiology of

lactating rabbits 44

3.2.a. Hormone treatments 45

INDEX

3.2.b. Biostimulation methods (without hormonal treatments) 46

4. Metabolic status and body condition of lactating rabbits during

the postpartum period

4.1. Total serum protein 50

4.2. Non-esterified fatty acids (NEFA) 51

4.3. Glucose 52

5. Objectives 53

6. References 54

CHAPTER 2ː BODY RESERVE AND OVARIAN PERFORMANC IN

PRIMIPAROUS LACTATING RABBIT DOES SUBMITTED TO EARLY

WEANING AS A STRATEGY TO DECREASE ENERGY DEFICIT

1. Introduction 75

2. Materials and methods 77

3. Results 83

4. Discussion 88

5. References 93

CHAPTER 3ː METABOLIC AND REPRODUCTIVE STATUS ARE NOT

IMPROVED FROM 11 TO 25 DAY POST- PARTUM IN NON WEANED

PRIMIPAROUS RABBIT

1. Introduction 99

2. Materials and methods 101

3. Results 107

4. Discussion 111

INDEX

6. References 116

CHAPTER 4ː GENERAL DISCUSSIONAND CONCLUSIONS

1. General discussion 123

2. General Conclusions 130

3. References 131

III

ABBREVIATION LIST

ºC: degree Celsius.

*: P<0.05.

**: P<0.01.

***: P<0.001.

%: percentage.

AI: Artificial Insemination.

BC: body condition.

BIA: bioelectrical impedance analysis. cAMP: adenosine 3 ', 5'-cyclic monofofosfato.

CGs: cortical granules.

GnRH: gonadotropin-releasing hormone.

COCs: cumulus-oocyte complexes.

DAB: 3, 3'-diaminobenzidine.

DAG: diacylglycerol.

DAPI: 4, 6-diamino-2-phenylindole.

DNA: deoxyribonucleic acid. dpp: days post-partum. dUTPs: nucleotides marked.

E2: Oestradiol-17β. eCG: equine chorionic gonadotropin.

EGF: epidermal growth factor.

ABBREVIATION LIST

EIA: Enzyme immunoassay analysis.

FAO: Food and Agriculture Organization (United Nations).

FI: feed intake.

FSH: follicle stimulating hormone. g/dL: grams per deciliter

GnRH: gonadotropin-releasing hormone.

GV: germinal vesicle.

GVBD: germinal vesicle breakdown. hCG: human chorionic gonadotropin.

HPO: hypothalamus-pituitary-ovary axis.

IGF-I: insulin growth factor I. i.m.: intramuscular.

IU: international units. iv.: intravaginal.

Kg: Kilogram.

LBW: live body weight.

LH: luteinizing hormone.

MAPK: mitogen-activated protein kinase.

MI: metaphase I.

MII: metaphase II. mEq/ L: milliequivalents of solute per litre. mg: milligram. ml: milliliter.

MPF: meiosis promoting factor. mRNA: messenger ribonucleic acid.

ABBREVIATION LIST

NEB: negative energy balance.

NEFA: non-esterified fatty acids. ng/ mL: nanograms per millilitre.

P: probability.

P4: Progesterone.

PBS: phosphate buffer solution.

PGF2 α: prostaglandin F2α.

PKC: protein kinase C. pg/ mL: picograms per millilitre.

PRL: Prolactin.

PRL-R: Prolactin receptor.

RIA: radioimmunoassay analysis.

SEM: standard error of the mean.

TUNEL: DNA labeling at the 3 'terminal deoxynucleotidyl mediated by the enzyme

transferase.

μg/ mL: microgram per milliliter.

μm: micrometer.

RESUMEN

El objetivo general de esta Tesis Doctoral ha sido tratar de mejorar los parámetros reproductivos de las conejas primíparas lactantes, empleando dos métodos de manejo (destete temprano y extensificación del ritmo reproductivo), que están directamente relacionados con su balance energético.

Para ello, se diseñaron 2 experimentos en este tipo de hembras. En el primero, se estudió el efecto del destete a 25 días post-parto (dpp) sobre la actividad ovárica y el metabolismo energético de las conejas una semana más tarde (32 dpp). Un total de 34 primíparas lactantes con 8 gazapos fueron distribuidas en tres grupos: 10 conejas se sacrificaron a los 25 dpp (grupo L25), 13 fueron destetadas a los 25 dpp y sacrificadas a los 32 dpp (grupo NL32), y 11 conejas no se destetaron y fueron sacrificadas a los 32 dpp (grupo L32). No se observaron diferencias significativas entre grupos en el peso corporal, el peso del ovario, ni en las concentraciones séricas de ácidos grasos no esterificados y de proteínas totales. A pesar de que el grupo NL32 presentó un bajo consumo de alimento (122 ± 23,5 g / día, p <0,001), su contenido corporal estimado de lípidos (16,9 ± 1,09%, P <0,008), proteínas (19,7 ± 0,07%, P <0,0001), y energía (1147

± 42,7 MJ / kg, p <0,006) fueron más elevados y las concentraciones séricas de glucosa

(158 ± 24,5 mg/dl, p <0,04) más bajas que en los grupos L25 (11,9 ± 1,3%, 18,5 ±

0,08%, 942 ± 51,3 MJ/kg y 212 ± 27,9 mg/dl) y L32 (13,4 ± 1,03%, 18,5 ± 0,1%, 993 ±

40,4 MJ/kg y 259 ± 29,5 mg/dl), respectivamente. En el grupo L25 se observó un menor número medio de folículos ≥ 1 mm en la superficie ovárica en comparación con los grupos NL32 y L32 (12,7 ± 1,5 vs. 18,0 ± 1,45 y 17,6 ± 1,67, p <0,05). La población folicular ovárica en las secciones histológicas y la inmunolocalización de los receptores de prolactina fueron similares en todos los grupos. En el grupo L25, tanto la maduración

RESUMEN nuclear de oocitos, medida en términos de tasas alcanzadas de Metafase II (67,0 vs. 79,7 y 78,3%, P <0.05) y la maduración citoplasmática, medida por el porcentaje de gránulos corticales (GC) total o parcialmente migrados en los oocitos, fueron significativamente menores que en los grupos NL32 y L32 (16,0 vs 38,3 y 60,0%, P <0.05).

En conclusión, a pesar de que el destete precoz a 25 dpp pareció mejorar las reservas de energía de las conejas primíparas, este hecho no se reflejó claramente a nivel ovárico a los 32 dpp y fue similar independientemente del destete, por lo que éste

último podría llevarse a cabo más tarde.

En el segundo experimento, se compararon dos ritmos reproductivos. Se utilizaron un total de 48 conejas primíparas lactantes con 8 gazapos que se asignaron al azar en dos grupos experimentales: a) lactantes sacrificadas a comienzos del post-parto (11 dpp) de acuerdo a un ritmo semi-intensivo (n = 24), y b) lactantes sacrificadas al final del período post-parto (25 dpp) de acuerdo con un ritmo más extensivo (n = 24). En ellas, se estudió el peso vivo, la composición corporal estimada, parámetros metabólicos y endocrinos (estradiol y progesterona) y características ováricas como la población folicular y la tasa de atresia, así como la maduración nuclear y citoplásmica de los oocitos. En este estudio, el peso vivo, el contenido de energía corporal, los depósitos grasos y los ácidos grasos no esterificados disminuyeron a lo largo del post-parto con respecto al momento del parto (P <0,05). Las concentraciones séricas de proteínas y glucosa aumentaron en el mismo periodo post-parto (P <0,05). Se observaron similares niveles de estradiol y progesterona en ambos ritmos, así como una población folicular, tasas de maduración nuclear (tasa de oocitos en metafase II) y citoplasmática

(porcentaje de oocitos con gránulos corticales migrados), similares en ambos momentos

RESUMEN del post-parto. Sin embargo, el número de folículos preovulatorios en la superficie ovárica fue menor (P <0,05) y la tasa de atresia tendió a ser mayor con un porcentaje también menor de folículos sanos (P <0,1) en los ovarios de las hembras sometidas al ritmo extensivo.

En conclusión, al final del post-parto (25 días), las conejas primíparas sin destetar muestran un deterioro de sus reservas corporales, de sus parámetros metabólicos séricos y de la calidad de sus oocitos; incluso se ha observado una ligera influencia negativa en el desarrollo de sus folículos ováricos. Por esta razón, se considera que en las conejas primíparas lactantes el manejo reproductivo extensivo (25 dpp) no presenta ninguna ventaja en comparación con el semi-intensivo (11 dpp).

A la vista de los resultados de estos dos experimentos, podemos decir que ni el destete temprano, ni la extensificación del ritmo reproductivo han conseguido una mejora en los parámetros reproductivos de una hembra primípara. Por ello, son necesarios más estudios sobre el estado metabólico de la coneja primípara lactante para conseguir métodos o estrategias que lo mejoren y tengan consecuencias directas sobre la actividad reproductiva y sobre su éxito productivo.

SUMMARY

The general aim of this Thesis was to study two management methods (early weaning and extensive reproductive rhythm) linked to the energy balance of the primiparous rabbit does to improve their reproductive performance.

In this sense, 2 experiments were conducted using this kind of females. In the first experiment, the effect of weaning at 25 days post-partum (dpp) on ovarian activity and energetic metabolism one week later (32 dpp) was studied. A total of 34 primiparous lactating rabbit does were used and distributed among three groups: 10 does euthanized at 25 dpp (group L25), 13 does weaned at 25 dpp and euthanized at 32 dpp (group NL32), and 11 non weaned does euthanized at 32 dpp (group L32). No significant differences were observed in live body weight, ovary weight, serum non esterified fatty acids (NEFA) and total protein concentration among groups. Although

NL32 does had a low feed intake (122±23.5 g/Day; P < 0.001), their estimated lipids

(16.9±1.09%, P < 0.008), protein (19.7±0.07%, P < 0.0001), and energy (1147±42.7

MJ/kg, P < 0.006) body contents were higher and their serum glucose concentrations

(158±24.5 mg/dl, P < 0.04) were lower compared to L25 does (11.9±1.3%, 18.5±0.08%,

942±51.3 MJ/kg and 212±27.9 mg/dl) and L32 does (13.4±1.03%, 18.5±0.1%,

993±40.4 MJ/kg and 259±29.5 mg/dl, respectively). A lower number of follicles ≥1mm was observed compared to NL32 and L32 groups (12.7±1.5 vs. 18.0±1.45 and 17.6

±1.67; P < 0.05) in the ovarian surface of L25 does. Follicular population in the histological ovarian sections and immunolocalization of prolactin receptor were similar in all groups. In group L25, both nuclear maturation of oocytes in terms of Metaphase II rate (67.0 vs. 79.7 and 78.3%; P < 0.05) and cytoplasmic maturation measured by

SUMMARY percentage of cortical granules (CG), totally or partially migrated in oocytes were significantly lower than in groups NL32 and L32 (16.0 vs. 38.3 and 60.0%; P < 0.05).

Consequently, a higher rate of oocytes with non-migrated CGs was found in group L25 than in groups NL32 and L32 (76.0 vs. 46.8 and 33.3%; P < 0.05).

In conclusion, even though early weaning at 25 dpp seemed to improve body energy stored in primiparous does, this fact was not well reflected on the ovarian status at 32 dpp, which was similar regardless of weaning time.

In the second experiment, two reproductive rhythms were compared. A total of

48 primiparous Californian x New Zealand White rabbit does suckling 8 kits were randomly allocated in two experimental groups: a) lactating does euthanized at early post-partum period (11 dpp) according to a semi-intensive rhythm (n = 24), and b) lactating does euthanized on later post-partum period (25 dpp) according to a more extensive rhythm (n = 24). Live weight, estimated body composition, serum metabolic and endocrine parameters (oestradiol and progesterone concentrations) and ovarian features like follicle population and atresia rate, and oocyte maturation were studied.

Live weight, body energy content, lipid depots and serum non esterified fatty acids

(NEFA) concentrations diminished from parturition time to post-partum period (P <

0.05). In addition, serum protein and glucose concentrations increased along post- partum time (P < 0.05). Similar oestradiol and progesterone levels were shown in rhythms as well as similar follicle population and nuclear and cytoplasmic maturation rates measured as metaphase II and cortical granule migration, respectively in both post- partum times. However, number of preovulatory follicles on the ovarian surface was

SUMMARY lower (P < 0.05) and atresia rate tended to be higher with also lower percentage of healthy follicles (P < 0.1) in ovaries of females of extensive group.

In conclusion, primiparous non-weaned rabbits does at late post-partum time (25 days), Did no show any improvement regarding body reserves, serum metabolic parameters and oocyte quality; even a slight negative influence has been observed in the development of their ovarian follicles. Thus this reproductive management does not present any advantage compared to earlier post-partum (11 days) reproductive rhythm.

In summary, according to the obtained results from these two experiments, we can say that the application of early weaning and the extensive rhythms did not achieve an improvement in the reproductive performance of primiparous does. Thus, it is necessary to conduct more studies about the metabolic status of the primiparous lactating doe to achieve strategies in order to improve it and consequently, to improve the reproductive activity and their productive success.

CHAPTER 1

LITERATURE REVIEW

AND OBJECTIVES

CHAPTER 1

1. Generalities

Rabbit does have particular reproductive characteristics, one of which is that not showing post-partum anestrus. In the intensive production of these species this feature is exploited to short parturition-mating intervals as much as possible. Thus, in this way, pregnancy and lactation overlapping for extended periods of time. In return, this requires very high energy requirements even more drastic in primiparous lactating rabbits, since in addition, these animals are growing and are unable to consume enough amount of food to meet their energy needs. This leads to a negative energy balance and a deterioration in body condition, which generates a sharp decline in fertility (around

40%), increasing the parturition intervals and the number of services per conception

(Xiccato, 1996, Pascual et al., 1998; Bolet and Fortun-Lamothe, 2002 and Rebollar et al., 2006a), all of which causes major economic losses on the rabbit farm. Therefore, knowing what happens in the first deliveries of the rabbit, which is one of the periods critical in the production performance of these species are extremely interesting for establish appropriate management guidelines.

2. Rabbit reproductive physiology

Rabbits have absence of a defined and regular estrous cycle since the ovulation

is induced by coitus rather than by the high concentration of estrogens. Coitus

generates a neuroendocrine reflex which stimulates the release of gonadotropin-

releasing hormone (GnRH) by the hypothalamus (Kaynard et al., 1990) resulting in

preovulatory discharge of luteinizing hormone (LH) which triggers oocyte maturation

and ovulation (Mills and Gerardot, 1984 and Pau et al., 2000).

CHAPTER 1

2.1. Oogenesis and follicular development

In most mammals, oogenesis begins during embryonic life and transformation of oogonia (diploid cell) in oocytes (haploid cell) is complete before birth (Fair, 2003). In the rabbit doe, the oogonias begin meiosis and become oocytes during the first 10 days of life (Peters et al., 1965 and Hutt et al., 2006). In the third week of age, the ovary shows primordial follicles and developing follicles (Gondos, 1969 and Hutt et al.,

2006), whereas antral follicles are first detected from the 12th week of life (Wolgemuth et al., 1984 and Hutt et al., 2006). Thus, in the first weeks after birth, the female has the endowment of oocytes available for the rest of her reproductive life. From here, there are waves of follicular growth (folliculogenesis) and regression (atresia) steadily, although the oocyte nucleus remains arrested in diplotene stage of prophase of the first meiotic division, nuclear stage that remains until a few hours before ovulation (Sirard,

2001).

However, during folliculogenesis, the molecular machinery of the oocyte is active. The nucleolus has a structure that reflects fibrogranular synthetic activity of the oocyte during growth (Sutovsky et al., 1993). During this period, the ovary synthesizes and stores large amounts of messenger ribonucleic acid (mRNA) and protein increasing their size (Motlik et al., 1989). It also increases the number of mitochondria, of cortical granules and the cytoskeleton, through which are distributed organelles (Ferreira et al.,

2009). In the rabbit, as stated by Jelínková et al. (1994), the majority of follicles less than 1 mm in diameter contains not competent oocytes. Therefore, the competence of the oocyte increases, usually with its size and the follicular diameter (Kruip and

Dieleman, 1982 and Jelínková et al., 1994). In fact, the results of in vitro maturation are

CHAPTER 1 better when using those oocytes found within 1 mm follicles and makes a selection themselves based on morphological criteria of homogeneity of the cytoplasm of the oocyte and a compact cluster. The denuded oocytes or with signs of vacuolization in the cytoplasm, cytolysis, necrosis or loss of spherical shape are considered as degenerate, and,therefore, not suitable for in vitro maturation (Jelínková et al., 1994 and Lorenzo et al., 1996).

In vivo conditions, for the correct development of both the oocyte and the cells that around during folliculogenesis and also during the subsequent maturation process, are essential numerous groove joints or joints "gap" formed by proteins transmembrane called connexins (Kidder and Mhawi, 2002 and Kennedy et al., 2003). Through of them, there is a bidirectional exchange of many signals such as: nutrients, metabolic precursors, hormones and growth factors (Sutton et al., 2003). They exert both positive and negative stimuli between the oocyte and cumulus cells (Van der Hurk and Zhao,

2005).

However, the precise regulation of folliculogenesis and follicle survival mechanisms and the in vivo oocyte is not fully known in the rabbit. In mammals, during folliculogenesis, follicles can be defined in five morphological stages which turn can be grouped in relation to the moment that forms the follicular antrum: preantral follicles

(primary and secondary) and antral (tertiary and preovulatory) (Figure 1):

 Primary follicle. The follicle has a diameter about 100-120 microns (Pincus and

Enzmann, 1935 and Hutt et al., 2006). Flat follicular cells surrounding the oocyte

CHAPTER 1

are transformed into a layer of cuboidal cells. The oocyte of the rabbit presents a

strong growth reaching a diameter.

 Preantral or secondary follicle. The follicle is about 200 microns in average

(Pincus and Enzmann, 1935). It consists of an oocyte that reaches almost its

maximum size (80 - 104 microns), with two or more layers of granulosa cells that

rapidly multiply and begins to develop the theca cells and vasculature (Kranzfelder

et al., 1984 and Macchiarelli et al., 1992).

 Antral follicle or tertiary. These follicles in the rabbits have a diameter greater

than 200-250 microns according to different authors (Fleming et al., 1984;

Kranzfelder et al., 1984; Jelínková et al., 1994 and Hutt et al., 2006). Some areas

appear with a follicular fluid inside produced by follicular cells, forming a follicular

cavity called the antrum. The oocyte grows to about 133-135 microns (Jelínková et

al., 1994) and has six to nine layers of granulosa cells.

A B C

Figure 1. Categorization of follicles in histological ovarian sections. A) Primary

follicles. B) Preantral follicles. C) Antral follicles. (From Rebollar et al., 2008).

CHAPTER 1

In the adult rabbit, the average time it would take a primary follicle to reach the

size preovulatory would be 97 days (Mariana et al., 1989). If there is no mating, the

waves of follicular development and regression are constant being the usual dynamics

of the ovary. These are developed and returning at intervals around 10 days (Hill and

White, 1933) coinciding with follicular size ranging from 450 microns, medium-antral

follicles, until they reach the preovulatory stage (Diaz et al., 1987). This means that

the cycles partially overlap each 4-6 days. The action of both follicle stimulating

hormone FSH (Follicle Stimulating Hormone) and LH (luteinizing hormone) is

essential for the development of such antral follicles. Both gonadotropins stimulate

ovarian steroidogenesis through their receptors. Granulosa cells express receptors for

FSH and have aromatase activity, an enzyme that catalyzes the synthesis of 17-β

estradiol from the androstenedione and testosterone from the theca (for LH receptor)

(Erickson and Ryan, 1975 and 1976). Therefore, the levels of normal 17-β estradiol

intrafollicular significantly increase with increasing the size of the healthy antral

follicles (Lefèvre and Caillol, 1978). In turn, the preovulatury peak of the LH causes

luteinization of the granulosa cells of preovulatory follicles and they begin to

synthesize predominantly progesterone instead of estradiol (Van der Hurk and Zao,

2005). Taking note that the cumulus cells are essential for the oocyte transcriptional

activity during folliculogenesis (Eppig, 2001), as well as for growth and maturation

(Lorenzo et al., 1996). Altered levels of estradiol and progesterone in the preovulatory

follicle during the postpartum period may affect these processes (Beker et al., 2002).

In particular, during the postpartum period, Diaz et al. (1987) noted that there is a first follicular wave in late pregnancy, coinciding with decreasing levels of circulating progesterone on days 29 and 30. These waves reach their peak in about 3 and 9 days

CHAPTER 1 post-partum (Díaz et al., 1987 and Ubilla and Rebollar, 1995) and at the end of lactation being related to the increased production of 17-β estradiol in the follicle and sexual receptivity (Lefèvre and Caillol, 1978 and Molina et al., 1986). However, a plasma level of 17-β estradiol given can not accurately predict the sexual receptivity level in this species (Rebollar et al., 1992), due to its extreme individual variability especially during lactation (Ubilla and Rebollar, 1995). In this sense, the inhibitory action of prolactin on follicular development could be determined by their interference on steroidogenesis induced by FSH in the follicle, or by direct depression of FSH secretion in the pituitary

(Bhattacharya et al., 2007).

2.2. Oocyte maturation

The oocyte maturation refers to two distinct biological events: a) the nuclear maturation, determined by the initial chromatin modifications, b) the cytoplasmic maturation, among other processes, completed with the migration of cortical granules

(CG) to the periphery of the oocyte to prevent polyspermy (Ducibella et al., 1993 and

Hoodbhoy and Talbot, 1994). Both processes are the culmination of the progressive oocyte development from the onset of folliculogenesis. (Hyttel et al., 1997).

2.2.1. Nuclear maturation

The step prior to ovulation is the reduction of genetic material to the half through the process of meiosis, giving rise to two daughter cells haploid: the oocyte itself, which retains nearly all the cytoplasm, and the polar body that contains practically only the chromosomes. As mentioned earlier, during oogenesis, the oocyte

CHAPTER 1 nucleus remains molecularly quiescent but active, in the diplotene stage of prophase I.

This phase is characterized by the presence of a configuration prominent nuclear called germinal vesicle (GV) (Sutovsky et al., 1993). The peak preovulatory LH triggers the resumption of meiosis due to loss of unions "gap" between the cumulus and the oocyte, as a result of the expansion of the former. These results in decreased levels of cAMP, which triggers the activation of certain protein kinase oocyte, such as: the meiosis promoting factor (MPF, Maturation Promoting Factor) and the mitogen-activated protein kinase (MAPK, Mitogen-activated protein kinases) which induce the resumption and progression of meiosis (Liang et al., 2007). It is known that this process is also triggered spontaneously when the oocyte is isolated from the follicle (Pincus and

Enzmann, 1935; Edwards, 1965 and Nicosia et al., 1975). The resumption of meiosis is identified by the breaking of the nuclear membrane of the GV, the disappearance of the nucleoli and condensation of chromosomes that are aligned in the metaphase plate

(metaphase I). Then, separate the pairs of chromosomes counterparts and meiosis progresses to metaphase stage of the second division meiotic (metaphase II) accompanied by the extrusion of first polar body (Deker, 2005). This period is known as the oocyte nuclear maturation (Figure 2)

The oocyte remains in metaphase II stage in the oviduct until fertilization occurs. At this time meiosis is completed producing extrusion of the second polar corpuscle.

CHAPTER 1

Figure 2. Schematic representation of the overall process of oocyte meiotic maturation in mammals. (From www.epigenetics.co.kr / oocyte.gif).

In the rabbit, the process of chromatin condensation occurs at the end of oocyte growth period before it restarts meiosis (Motlik et al., 1989 and Sutovsky et al., 1993).

Condensed chromosomes can be observed to a lesser or greater degree inside the GV with the size of the follicle in which it arises. Thus, the oocytes from follicles with a size around 1 mm in diameter have chromosomes bivalent inside the GV (Jelínková et al., 1994).

2.2.2. Cytoplasmic maturation

Cytoplasm of the oocyte plays a crucial role in the assembly of machinery for the production of metabolic energy. This energy is needed to perform cellular functions during maturation, fertilization and early embryonic development until the activation of

CHAPTER 1 embryonic genome. Cytoplasmic maturation is a process involving post-transcriptional modifications of mRNAs accumulated during the oogenesis and the relocation and modification of some organelles of the oocyte (Ferreira et al., 2009).

Protein synthesis is crucial in periods of development (Van Blerkom and

McGaughey, 1978). During maturation, it increases approximately three times from the stage of GVBD to MI. When the oocyte reaches MII, basal levels are again observed.

(Tomek et al., 2002). The major transcripts produced, encode proteins cell cycle regulators such as, among others, the meiosis promoting factor (MPF) and the mitogen- activated protein kinase (MAPK) (Liang et al., 2007), and proteins that make up the system antioxidant enzyme such as glutathione and peroxidases, protecting the oocyte against the oxygen free radicals generated by mitochondrial metabolism (Ali et al.,

2003).

At the ultrastructural level has been observed that during maturation increases the number of copies of mitochondrial DNA, and mitochondria travel along the microtubule cytoskeleton from the periphery to the central area of the oocyte. Lipid content increases and reduces the Golgi apparatus. The endoplasmic reticulum distributed throughout the cytoplasm is accumulated in the cortical regions. Among other functions, is responsible for regulating the storage and release of Ca2+ intracellular. The cortical granules (CG) are spherical organelles of small size (between

100 and 200 nm in diameter) that are present exclusively in the cytoplasm of oocytes

(Gulyas, 1974a and 1974b). They are formed from the Golgi apparatus during folliculogenesis (Hoodbhoy and Talbot, 1994). Are delimited by a single membrane containing glycoprotein residues (Gordon and Dandekar, 1976) and mainly proteases

CHAPTER 1

(Hoodbhoy and Talbot, 1994). During fertilization, the cortical endoplasmic reticulum releases large amount of Ca2+ involved in the exocytosis of cortical granule contents

(Ferreira et al., 2009). Microfilaments and microtubules that make up the cytoskeleton are involved in peripheral migration processes, in the separation of chromosomes, in the establishment of polarity in the oocyte, the extrusion of the first and second polar body and migration and exocytosis of cortical granules (Sun and Schatten, 2006).

The detection of CG can be performed by electron microscopy or fluorescence.

The use of lectins linked to fluorochromes for its location was used primarily in the mouse (Ducibella et al., 1988) and hamster (Cherr et al., 1988) and later in domestic species (Yoshida, 1993; Hosoe and Shioya, 1997; Carneiro et al., 2002 and Velilla et al., 2004). Lectins are substances that have the property selectively bind to certain radical glucidic, so they are a tool useful for detecting glucose-conjugated compounds found in the membrane of the CG. In particular, the CG of rabbit oocytes are selectively detected by the LCA lectin (lens culinaris agglutinin) that binds specifically to the radical present the monosaccharide α-D-mannose (Hoodboy and Talbot, 1994). During oocyte maturation, the cortical granules migrate to the periphery (Gulyas, 1974a and

1974b) and are distributed in a monolayer beneath the plasma membrane (Wang et al.,

1997a).

The main function of cortical granules lies in its ability to block polyspermy during the fertilization of the oocyte by means the cortical reaction (Fraser et al., 1972 and Fléchon et al., 1975). This is generated in response to the fusion of sperm to the oolema mediated by a G protein, which triggers the activation of two secondary messengers: inositol triphosphate (IP3) and diacylglycerol (DAG). This results in an

CHAPTER 1 increase in intracellular Ca2+ from the endoplasmic reticulum and activation of the protein kinase C (PKC) leading to exocytosis of granule contents to the cortical perivitelline space, modifying the structure of the zona pellucida and inactivating specific receptors that recognize and bind sperm (Ducibella et al., 1993 and Wassarman,

1999). Calmodulin-dependent kinase II is another molecule involved in this process

(Abbott and Ducibella, 2001) and in the rabbit meiotic maturation (Henry et al., 1997).

In this way, and quickly, there is the "Reaction zone" in the pellucida zone which prevents the binding of new sperm and thus, blocks the possibility of multiple penetrations (polyspermy). This locking mechanism is highly conserved among species.

The penetration of several sperm into the oocyte results in the formation of supernumerary centrioles and aberrant division processes generating non-viable embryos in the early mitotic divisions. In particular, the CG rabbit oocytes release large amount of mannose or glucose residues (Gordon et al., 1975). This would generate a large number of negative charges in the perivitelline space, which leads to a modification of oolema and a repulsive force to sperm (Cooper and Bedford, 1971), which could contribute to block polyspermy in this species (Braden and Austin, 1954 and Overstreet and Bedford, 1974).

In vitro, the causes of polyspermy have been attributed to dysfunction of exocytosis the CG, a delay in peripheral migration or irregular distribution of these structures (Gulyas, 1974a and 1974b). Therefore, the distribution of CG has been used as a criterion for evaluating the degree of maturity reached by the oocyte cytoplasm in different species and consequently to predict the subsequent fertilization success (Wang et al., 1997b).

CHAPTER 1

During oocyte cytoplasmic maturation, we can find the following distributions of

CG (Hosoe and Shioya, 1997; Carneiro et al., 2002 and Velilla et al., 2004) (Image 4):

 Peripheral distribution. The CG are placed just below the plasma membrane. This

pattern would correspond to a cytoplasmic maturation oocyte proper.

 Cortical distribution. The CG are in the cortical region separate from the plasma

membrane. This configuration corresponds to oocytes that have initiated CG

migration, but this is incomplete.

 Homogeneous / Diffuse distribution. The CG are distributed throughout the entire

cytoplasm space. This distribution corresponds to immature cytoplasmic oocytes.

 Non homogeneous distribution. The CG are abnormally distributed following any

type of distribution pattern. CG aggregates appear in the cytoplasm. This

configuration could be compatible with degenerated or low quality oocytes (Gulyas,

1974a and 1974b).

Figure 3. Schematic representation of the main patterns of distribution of the granules in the oocyte cortex. A) Peripheral. B) Cortical. C) Diffuse / homogeneous. D) No homogeneous / abnormal. (From Arias-Álvarez, 2009).

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The migration of CG is correlated with the nuclear maturation process (Velilla et al., 2004). The failure of any of these during oocyte maturation results in the impossibility of a successful fertilization and posterior embryonic development (Plachot and Crozet, 1992; Yang et al., 1998 and Sirard, 2001).

2.3. Steroidogenesis in cumulus-oocyte complexes (COC)

As previously described, during follicular development FSH stimulates aromatization of testosterone in granulosa cells of follicles. This effect of FSH on estradiol production in granulosa cells has also been demonstrated in vitro (Schreiber et al., 1980 and Zachow and Magoffin, 1997). For its part, the preovulatory discharge of

LH induces differentiation and luteinization of the granulosa and cumulus cells in preovulatory follicles. This process generates a change in the pattern expression of genes essential for steroidogenesis. Some authors have shown even that cells acquire the ability to secrete spontaneously progesterone before the LH peak in isolated preovulatory follicle (Hillensjo et al., 1981; Vanderhyden and McDonald, 1998;

Channing et al., 1981 and Veldhuis et al., 1982). These facts suggest that presence of steroids in follicular fluid during the periovulatory period may have an effect on oocyte maturation in vivo (McNatty, 1978 and Ainsworth et al., 1980), and an impaired steroid balance has been shown to result in worse rates of maturation (Andersen, 1993).

During in vitro maturation, cumulus-oocyte complexes (COC) also have the ability to secrete steroids, a phenomenon observed in several species (woman: Chian et al., 1999; cow: Mingotti et al., 1995; sow: Dode and Graves, 2002; sheep: Shirazi and

Moalemian, 2007) including the rabbit (Lorenzo et al., 1997). The oocyte can, in turn,

CHAPTER 1 modular steroidogenic secretion in vitro through the secretion of factors not fully elucidated (Tsutsumi et al., 1980; Suzuki et al., 1983; Lucidi et al., 2003 and Shirazi and

Moalemian, 2007). In this sense, both estradiol and progesterone exert their local action on oocyte maturation through their specific receptors located in the cumulus cells and oocyte, in an autocrine or paracrine way (Naess, 1981; Park and May, 1991 and

Danforth, 1995).

2.4. Follicular atresia

Follicular atresia (Greek term that means a = no, tresos = perforation) is a degenerative process by which ovarian follicles are eliminated during their growth process physiologically by apoptosis or programmed cell death (Tilly et al., 1991).

At the biochemical level, in cell death by apoptosis there is a genetic program that is essential. The balance between the signals pro-and anti-apoptotic, determines the fate of the cell. These phenomena can be detected morphologically, since cells in the process of apoptosis have changes in to the cytoplasm and the nucleus that were first described by Kerr et al. (1972). Whatever the mechanism of activation of the apoptotic cascade, eventually triggers the activation of effector caspases such as Caspase 3, which is responsible for initiating signals that promote the release of Caspase-activated DNase. providing one of the biochemical features of this process (Hussein, 2005).

The nuclear membrane and the nucleus disintegrate into dense spherical fragments, also activate endonucleases dependent Ca2 + and Mg2 +, which degrade the

DNA into fragments of 180 to 200 base pairs, approximately (Peitsch et al., 1993).

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DNA fragmentation is an important indicator of the existence of the apoptotic process and is a specific marker for detection (Hussein, 2005). In the cytoplasm, the organelles are preserved but their organization and location is altered. However, the changes that occur in mitochondria are imperceptible from a structural point of view.

Subsequently, the plasma membrane ruptures and the cell shows vesicles form inside containing organelles and DNA, which are called apoptotic bodies. They are engulfed by neighbouring cells (Henson et al., 2001). In contrast to necrosis, death by apoptosis does not show the phenomenon of inflammation or if present, is almost imperceptible.

Most studies provide evidence for the importance of apoptosis in the ovary are focused on specific degeneration that occurs in granulosa cells (Palumbo and Yeh, 1994 and Matikainen et al., 2001). The granulosa cells of follicles positive for TUNEL

(Figure 4). have the biochemical, morphological and ultra-structural changes involved in the process of apoptosis, as described previously (Hussein, 2005). In the ovary, follicular apoptosis is a process that occurs continuously, with the atresia of most of the follicles (Tilly et al., 1991). The waves of follicular growth and regression constants in the rabbit, determined that while an antral cycle follicles are preovulatory, prior to regression (atresia), start the growth of follicles in the next wave. Therefore, the process of follicular antral atresia population is constant and high in this specie (Kranzfelder et al., 1984). In the phenomena of follicular development and atresia are involved local and systemic regulatory factors that inhibit or promote the appearance of apoptosis.

CHAPTER 1

1 2 3

Figure 4. Follicular apoptosis index in fixed ovaries of 14-weeks prepuberal females by

TUNEL assay (In Situ Cell Death Detection Kit, POD, Roche Diagnostics GmbH).

1) Late atretic follicle (>50% of apoptotic cells). 2) Early atretic follicle (<50% of apoptotic cells). 3) Healthy follicles. (From Garcia-Garcia et al., 2011).

Recent studies with in vitro granulosa cells of the rabbit, show that FSH promotes not only the development of antral follicles but also reduces the appearance of apoptosis (Maillet et al ., 2002 and 2003). By contrast, post-coital LH or human chorionic gonadotropin (hCG) contribute to the detachment in the size of dominant follicles screened for subordinates in the rabbit. According to Kranzfelder et al. (1984), those subordinate antral follicles with a diameter about 700 microns are atresiarian when the rabbits were given hCG. In other species like rats, it has been suggested that the susceptibility of antral follicles to atresia may be due to the acquisition of DNase, a phenomenon that occurs in the preantral population (Boone et al., 1995). However, the mechanisms of dominance and how they influence the final number of preovulatory follicles are not fully explained.

In addition, alterations in plasma concentrations of metabolic and hormonal factors associated with negative energy balance, also induce apoptosis processes in

CHAPTER 1 granulosa cells, either directly (Vanholder et al., 2005 and Hamm et al., 2004) or indirectly by modulating steroidogenesis (Swain et al., 2004). In this sense, the follicles that have a high number of apoptotic granulosa cells secrete significantly less estradiol because these cells lose their aromatase activity (McNatty et al., 1984). These follicles contain oocytes with a reduced quality (Blondin and Sirard, 1995 and Han et al., 2006) which affects ultimately, fertility (Leroy et al., 2008). The oocyte in turn, appears to control the proliferation and differentiation of granulosa cells, their response to gonadotropins, steroidogenesis, differentiation of thecal cells and cumulus expansion during ovulation (Lorenzo et al., 1997 and Van der Hurk and Zhao, 2005). Therefore, oocyte degeneration is an event that induces apoptosis of granulosa cells and vice versa, resulting in the phenomenon of follicular atresia.

2.5. Oestradiol-17β (E2) and Progesterone (P4)

Rabbits do not have a well-defined oestrous cycle and they are often, erroneously, considered to be permanently in oestrous. Sexual receptivity can be evaluated more accurately by the behavioural test in the presence of a vasectomized buck rather than by the colour of the vulva and its turgidity (IRRG Guidelines, 2005).

The relationships between female sexual behaviour, sex steroid hormones, and colour and turgidity of the vulva have been investigated in rabbits throughout pregnancy, pseudopregnancy, and post-partum period (Stoufflet and Caillol, 1988 and Rodriguez and Ubilla, 1988). During pregnancy, no clear correlation was detected; moreover, the does were sexually receptive despite high blood progesterone and low oestrogen concentrations. In contrast, during pseudopregnancy, females with a white vulva never accepted mating and the number of those with a red vulva, being sexually receptive,

CHAPTER 1 gradually rose toward the end of pseudopregnancy when progesterone declined to basal levels. In the post-partum period, the does are highly receptive the first day after parturition and shortly (1-2 days) after weaning (Theau-Clément, 2000). However, great variability exists among individual rabbit females depending on parity, lactation stage, and other factors (Theau-Clément and Roustan, 1992). Whereas the effect of plasma progesterone on oestrus behaviour depends on whether the does are either pregnant or pseudopregnant, administration of progesterone to estradiol-treated rabbits consistently suppresses sexual receptivity. In pseudopregnant not-receptive females, estrogens were not detectable in peripheral serum, but ranged between 15-140 pg/ml in those classified as receptive (Caillol et al., 1983). However, the physiological role of sex steroid hormones and their receptors in modulating genital hemodynamics (responsible for the external appearance of the vulva in terms of colour and turgidity, smooth muscle contractility of the genital tract, neurotransmitter receptor expression in the hypo thalamic-pituitary axis, and, finally, sexual behaviour) still requires further investigation in rabbits. Surprisingly, the role of androgens in the oestrous behaviour of rabbits has not been explored, despite the fact that a substantial amount of estrogens may derive from peripheral conversion of adrenal androgens and from aromatization of testosterone in the brain (Roselli et al., 1997).

Results indicate that sexual receptive behavior of the doe is related to the presence of more large follicles on the ovary (Kermabon et al., 1994) and higher plasma concentration of estrogens (Rebollar et al., 1992). It is now well established that sexual receptivity of the doe at the time of AI greatly affects fertility (Theau-Clément and

Roustan, 1992 and Castellini and Lattaioli, 1999) and its components, ovulation frequency (Rodriguez and Ubilla, 1988) fertilization rate (Theau-Clément, 2001) as well

CHAPTER 1 as prolificacy resulting from ovulation rate, embryo and foetal survival (Theau-Clément and Roustan, 1992; Castellini and Lattaioli, 1999 and Theau-Clément, 2001).

Consequently, the productivity of receptive does is higher than that of non-receptive does (primiparous: 6.3 vs. 1.6 weaned rabbits/AI, multiparous: 7.8 vs. 2.9 weaned rabbits/AI (Theau-Clément, 2001). Morever, regarding the physiological status of does at the time of AI, lactating, non-receptive does have the worst reproductive performance following AI.

2.6. Ovulation and corpus luteum formation

As pointed out previously, ovulation is induced by coitus in the rabbit (Heape,

1905). The preovulatory LH that occurs as a result, peaking at between an hour and a half and two hours after treatment (Mills and Gerardot, 1984 and Pau et al., 2000).

Ovulation of oocytes occurs in both ovaries, at about 10-12 hours after preovulatory discharge of LH. Therefore, in this species, hormone treatments are now easily controllable with GnRH analogues in the case of performing AI procedures. After ovulation, granulosa and theca cells raises the corpus luteum producing progesterone.

This is not fully formed until after 6 days in the rabbit, and the highest levels of progesterone are reached within 12-13 days, declining progressively during the last days of gestation (Stoufflet and Caillol, 1988 and Rebollar et al., 1992). In turn, the presence of embryos in the uterus exerts an anti luteolític effect, as it prevents the production of prostaglandin F2α (PGF2a) by the endometrium, although it requires a minimum of two embryos implanted, so that the pregnancy could continue (Hafez, 1968).

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Introduction of rabbits on fixed days of the week has significantly improved the management, the human resources and reproductive performance in rabbit farms. Due to the physiological characteristics further mentioned of this species, it is necessary to apply methods to induce ovulation using the intramuscular administration of GnRH synthetic analogues at the time of AI (Theau Clément et al., 1990 and Rebollar et al.,

1992).

The administration of GnRH is the most reliable method for inducing ovulation because it allows for repeated treatments and does not induce antibody formation, in contrast to repeated injections of LH or hCG (Adams, 1961). Several different GnRH analogues have been used (gonadorelin, buserelin, leuprolide, triptorelin), and the standard AI technique includes an intramuscular injection at different dosages depending on the strength of the GnRH analogue (Rebollar et al., 1997; Theau-Clement et al., 1990 and Zapletal and Pavlik, 2008). GnRH analogues can be absorbed subcutaneously or by the mucous layers because they have small molecular weights because the gonadorelin is a decapeptide and buserelin a nonapeptide (Dal Bosco et al.,

2011).

On rabbit farms, GnRH is usually administered by the farmer, which carries the risk of misuse and an increased amount of time needed for each AI. The possibility of adding GnRH directly into the seminal dose with results similar to those obtained by i.m. injection would be beneficial for the farmer. According to Quintela et al. (2004), they compared an intramuscular (i.m.) injection of buserelin (0.8μg/doe) with 8 and

16μg/doe added to the insemination dose. The kindling rates were 82%, 56%, and 85% for each treatment, respectively. Viudes-de-Castro et al. (2007) compared the iv. effects

CHAPTER 1 of two synthetic GnRH analogues (buserelin and triptorelin) given at two dosages. He found the ovulation rate was very low (32.5%) in the negative control group (does inseminated with 0.5mL of non-supplemented extender) and was higher in the positive control females (1μg/doe buserelin i.m.) 97.8%. Using only 5μg/doe buserelin iv. achieved an ovulation response similar to the positive control group. Furthermore,

Vicente et al. (2008) found that supplemented semen with 10g/ml of buserelin acetate given to the iv. groups, regardless of the physiological status of females or the farm, presented lower pregnancy and kindling rates respect to control does (intramuscularly treated with 1g of buserelin acetate).

3. Reproductive Management Strategies

3.1. Effect of remating intervals on reproductive physiology of lactating rabbits

The reproductive rhythms applied in rabbit farms can be:

 Intensive Rhythm: AI is performed on day 4 post-partum (parturition interval:

about 35 days).

 Semi-intensive Rhythm: AI was performed 11 days post-partum (parturition

interval: about 42 days).

 Semi-extensive Rhythm: AI at 18 days postpartum (parturition interval: around 49

days).

 Extensive Rhythms: AI at 25 days post-partum (parturition interval: around 56

days) or AI at 32 days post-partum (parturition interval: about 64 days).

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In the European farms, the most widespread reproductive management is the semi-intensive rhythm, so the most of the females inseminated are lactating. In the rabbits, lactation not totally stops their ovarian activity, as there are waves of follicular growth during the post-partum period (Diaz et al., 1987 and Ubilla and Rebollar, 1995).

However, the AI in lactating females strongly committed fertility and prolificacy, especially in the second insemination, when the does are primiparous, lactating and still continue with its own growth, compared to non-lactating or with multiparous lactating rabbits does (Lamb et al., 1991; Rebollar et al., 2004 and 2006a). This leads us to believe that the negative effect of lactation on reproductive function is mediated by prolactin (Ubilla et al., 2000a and 2000b), but also by the marked energy deficit resulting from the milk production, growth needs of primiparous rabbit and overlap of lactation and gestation from 11 to 18 days postpartum (Xiccato et al, 2005). This generates significant economic losses due to inefficient reproductive exploitation of this group of animals.

Rabbit needs a good balanced nutrition scheme for efficient productivity because they fail to achieve their energy requirements despite mobilize part of their body reserves (Lamb et al., 1984; Partridge et al., 1986; Parigi-Bini et al., 1992; Xiccato et al., 1995; 1996 and Fortun-Lamothe et al., 1999). Therefore, a factor to take into account to improve the energy balance of these animals is choosing an appropriate reproductive rhythm that maximizes performance and improves productivity taking into account the physiological characteristics of does (Cervera et al., 1993 and Xiccato et al.,

2005).

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Lactating rabbits not synchronized and inseminated at around day 11 of lactation are more fertile than those inseminated 3 or 4 days post-partum (Rebollar et al., 1992).

The latter have a high sexual receptivity, which coincides with a wave of follicular growth (Diaz et al., 1987), but have a lower rate of ovulation and oocyte fertilized

(Torres et al., 1977 and Lamb et al., 1991). This may be due, among other causes that around day 4 of lactation females have a large number of pre-ovulatory follicles not responding to injection of GnRH and thus do not ovulate. In practice, it was found that increased production by use of these reproductive cycles has no advantages over the 11 days postpartum, as they generate energy depletion of does, increasing the replacement rate considerably, and decreasing the number of weaned rabbits. Thus, in a long-term study (Rebollar et al., 2006a and 2006b), which applied an intensive rhythm for nine cycles, it was observed that the average interval between births was 45 days, similar to what would be achieved with insemination on day 11 post-partum, which is 42 days.

Although the productivity of rabbits under semi-intensive rhythm is superior to intensive rhythms (Rebollar et al., 2009), the fertility of lactating primiparous rabbit does inseminated on the day 11 post-partum is still very low (50-60%) compared with that obtained in nulliparous and multiparous 80-90% (Castellini et al., 2003 and

Rebollar et al., 2009). This is because AI on day 11 post-partum is a protocol adapted to the production cycles, but it no takes into account the physiological peculiarities of primiparous rabbits, and that destabilize their energy reserves, reducing the quality of oocytes when they have multiple pregnancies (García-García et al., 2008). The replacement rate remains high (Rebollar et al., 2009), which means a decrease length of productive life breeding mothers, since many of them die before weaning their third litter (Cervera et al., 2006). Prolonging the life of the rabbits is desirable not only from

CHAPTER 1 the point of view of animal welfare but also to compensate for expenses caused by the female during breeding.

In this sense, extensive reproductive cycles, in which the female is inseminated after weaning, allow better recovery time not having to support the energy expenditure caused by growth and simultaneous gestation and lactation from day 11 postpartum

(Fortun-Lamothe, 2006). The females have to be inseminated in the negative band of the previous AI, who has passed their period of pseudopregnancy, i.e., on day 32 post- partum (11 days from calving to the AI, plus 21 days of the pseudopregnancy period).

At this time, prior to the AI, kits have already begun to feed solid feed and gradually reduced milk consumption, and can even be weaned. In fact, the insemination of multiparous does in advanced lactation periods improves reproductive parameters

(Velasco et al., 2008 and 2009). However, extensive rhythms always have been considered unprofitable, since it increases the parturition interval, although reproductive parameters, the number of rabbits and length of life production can improve (Xiccato et al., 2005 and Castellini et al., 2006). Even the alternation extensive - intensive rhythm

(rabbits inseminated on day 1 post-partum and post- weaning, respectively) appears to reduce the energy deficit and annual replacement rate mothers, when compared with a fixed semi-intensive rhythm (AI on day 11 forever post-partum) (Castellini et al., 2003).

3.2. Effect of estrus synchronization on reproductive physiology of lactating rabbits

Systematically the not synchronized lactating rabbits have fertility rates lower than the non-lactating (Rebollar et al., 2004 and 2006a). At the moment, there are a

CHAPTER 1 large number of interrelated factors that affect oocyte competence and fertility. To solve this problem, it is necessary to use various methods of estrus synchronization to stimulate follicular development and estradiol secretion that trigger a state of sexual receptivity favorable at the time of AI, for properly induce ovulation and improve reproductive parameters (Castellini, 1996; Rebollar et al., 2006a and 2006b).

3.2. a. Hormone treatments

The growth of antral follicles is gonadotropindependent, subjected to the contribution of FSH and LH. To stimulate ovarian response and follicular development, one of the most widely used techniques in multiparous rabbits during the postpartum period is the administration of equine chorionic gonadotropin (eCG). The eCG whose action is similar to the FSH (70%) and LH (30%) is administered in a single dose

(Siddiqui et al., 2002). In general, a dose of 25 IU of eCG 48 hours before AI stimulates follicular development in the latter stages without inducing superovulation, improving the fertility of primiparous and multiparous lactating rabbits up to 30% (Bourdillon et al., 1992 and Rebollar et al., 2006a). In multiparous rabbits, standard doses of eCG (25

IU) stimulated in vitro oocytes maturation compared with their lactating rabbits unstimulated (García-García et al., 2006a). However, other authors have described lower doses of eCG (5 to 20 IU), because stimulate development of a greater number of follicles, but in return, reduce early embryo development (Bonanno et al., 1990).

The repeated use of eCG in rabbits could lead to health problems and carcass residues. Moreover, it generates an immune response (Lebas et al., 1996), greatly decreasing their reproductive success (Rebollar et al., 2006a and 2006b), although this

CHAPTER 1 response is highly variable and individual. Sometimes there are significant improvements when there are fertility problems, as is the case of primiparous rabbits

(Rebollar et al., 2006a). Hormonal synchronization method could be replaced by nonhormonal methods resulted in health benefits (Alvariño et al., 1998; Rebollar et al.,

2006a and 2006b).

3.2. b. Biostimulation methods (without hormonal treatments)

The use of exogenous products (hormones, antibiotics,…etc) goes against the popular perception of the consumer. During the 6th World Rabbit Congress (Castellini,

1996) mentioned that the European Community policy might impose a restriction on the use of hormones (gonadotropins) in relation to their residues in meat, animal welfare and the desire of preserve a "natural" image of meat. A similar trend concerns of the antibiotics in order to counteract antibiotic resistance.

For these reasons, over these last years, important work has been done to set up alternative methods which do not require the use of hormones, in order to increase sexual receptivity at the moment of insemination and consequently the productivity of rabbit does, These are called “biostimulation” methods.

Up to date, several approaches have been tried such as: animal manipulation

(doe-gathering), dam-litter separation, feeding programs, photo period, and buck effects.

These methods were sometimes drawn from results obtained in other zootechnical species.

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In the case of lactating rabbits, one of the most commonly used methods is the dam-litter separation for a short period of time prior to the AI (Alvariño et al., 1998).

This operation generates a reduction in serum prolactin, thus decreasing the inhibitory effect exerted by this hormone on reproduction. In this way, improves the secretion of

FSH and related mechanisms with the steroidogenic ovarian follicular activity increasing sexual receptivity (Ubilla et al., 2000a; 2000b and Rebollar et al., 2008).

A temporary separation from the litter for 48 hours causes a reduction in weight of kits at weaning (Bonanno et al. 2000 and 2004) and increased mortality (Rebollar et al., 2004 and 2006a). Taking into that the rabbits are unique in that only nurse their kits once daily for a few minutes and preferably in the early hours of the morning, the tendency, therefore, is to use litter separations shorter than 24 hours, to avoid the rabbit miss any nursing or just delay it for a few hours until the time of AI. Previous studies on the hormonal profiles rabbit show that a temporary weaning for 24 hours is sufficient to induce estrus and enhance fertility in lactating rabbits (Castellini et al., 1998; Cano et al., 2005 and Rebollar et al., 2006a), especially during the first lactation (Maertens,

1998 and Bonanno et al., 2002).

In this sense, there is a great interest in early weaning technique that lies in the possibility of reducing the doe body energy deficit, by shortening the lactation length and prolonging the dry period. In does at their first, second and third parturition, reducing weaning age from 32 to 21 d of age improved body energy balance (from -

19% to 8% of the initial energy concentration) but was unable to achieve equilibrium

(Xiccato et al., 2004). Also in multiparous does, weaning at 25 d did not prevent body energy deficit (-8% of the initial energy content) while weaning at 21 d only provided a

CHAPTER 1 balance that approached equilibrium (3%) (Xiccato et al., 2005).

Scientific literature on the reproductive performance of does submitted to early weaning is scarce. A negative effect of early weaning on rabbit reproductive performance was al so recorded in terms of the lower number of kits born and kits born alive per litter in multiparous do es whose litters were weaned at 21 days of age

(Xiccato et al., 2004 and 2005). This result could be explained by the effects of weaning on metabolic and hormonal patterns which occurred at the time of foetus implantation from 7 to 11 days of pregnancy (Brecchia et al., 2005; Fortun-Lamothe,

2006). The occurrence of mastitis may also be observed in the field as a consequence of the abrupt interruption of lactation at 21-25 days. When early weaning (25 d) was associated with an early mating (4 d post-partum), higher prolificacy and litter size at weaning were observed in comparison with does subjected to a traditional system mating-weaning system (mating 11 days post-partum and weaning at 35 days of age)

(Nicodemus et al., 2002). These results cannot be c1early ascribed to weaning age or reproductive rhythm, however.

4. Metabolic status and body condition of lactating rabbits during the postpartum

period

Productivity levels of breeding does are comparable to those obtained in other domestic species. Thus, the rabbit milk production reaches 6.5 kg / lactation, for an average litter size of 8 rabbits (De Blas et al., 1995). On the other hand, the rabbit milk has a high concentration of nutrients, containing on average, 13.7% fat and 10.9%

CHAPTER 1 protein (De Blas et al., 1995). This indicates the great loss of energy of this animal at this stage.

In addition, as previously described intensification of production means that lactating mothers are inseminated shortly after the previous birth, Therefore, this management on the one hand, it not allow them to regain body reserves used in the previous pregnancy for fetal growth (Young, 1979), and secondly, it increases energy demand due to the new gestation overlapped with lactation. Therefore, although the reproductive capacity of the rabbit is one of the most highest of all domestic mammals, the use of such reproductive rhythms keeps it continually in a state of high nutritional needs which significantly reduce the reproductive outcome in this species (Parigi-Bini et al., 1992; Xiccato et al., 2004 and 2005).

This problem is further highlighted during the first lactation, in which the rabbit does not have a feed intake to cover multiparous females their energy requirements.

This leads them to mobilize its body reserves; in fact has been reported to mobilize about 52% of their fat reserves (ParigiBini et al., 1992; Xiccato et al., 1995 and Pascual et al., 2002). It is established that, during this period, a mother loses about 300 g of body fat and 10 MJ of energy (Forthun-Lamothe et al., 2002). On the contrary, it was observed that the extensive rhythms reduce the concentration of body water, improve the percentage of fat carcasses and energy balances come into balance at the time of the insemination (Xiccato et al., 2005).

The decrease in the nutritional and caloric negative energy balance during the generate postpartum body composition changes that are reflected in variations metabolic

CHAPTER 1 and hormonal blood levels (Parigi-Bini et al., 1990 and Pascual et al., 2002) negatively affecting at the time of AI, both sexual receptivity and fertility of the rabbit (Fortun-

Lamothe, 2006). The moments around ovulation are considered crucial and a complex group of hormones and metabolites are reported at the hypothalamic-pituitary and ovarian level. Some of them such as protein, non-esterified fatty acids (NEFA), or hormones related to body fat and the feed intake, such as leptin, are present in ovarian follicles of different species (mice, Kawamura et al., 2002; sow: Craig et al., 2004; cow and Leroy et al., 2005) and in the oviduct (mice: Harris et al., 2005) which could be influencing the follicular development in the competence of the oocyte and embryo survival (Sutton et al., 2003). In other species, there is increasing evidence that endocrine alterations and metabolic diseases that are triggered during post-partum as a result of negative energy balance significantly affect oocyte quality and embryo, these being the main factors that determine reproductive inefficiency animals during this period (Leroy et al., 2008).

4.1. Total serum protein

In regard to protein metabolism, total serum proteins concentration in rabbits is between 5.4 and 7.5 g/dl (Melillo, 2007). Factors such as age or gestation can alter these values. According to Deichmiller and Dixon (1960), when the rabbits are pregnant, serum proteins of animal reserves are depleted, reducing its baseline concentration and even leading to a marked hypoproteinemia in time of delivery. In addition, according to these authors, when the animal cannot perform well coprophagy for its advanced state of pregnancy significantly reduces the ingestion of a protein source very important that corresponds to 25-30% of dry matter percentage (Kupersmith, 1998). The

CHAPTER 1 hypoproteinemia also can be started by dehydration or chronic malnutrition (Melillo,

2007).

The permeability of the follicle to plasma proteins increases during follicular development, since it has been observed that there is a positive relationship between the size of follicle and the concentration of protein in it (Sutton et al., 2003). In particular, the albumin concentration in follicular fluid is higher than in plasma of women

(Velazquez et al., 1977). In this sense, in some species like the pig (Cia et al., 1998) have suggested that a decrease in the percentage of plasma protein affects considerably the reproductive capacity. This may be due, among other factors, to alterations in amino acid concentrations could be exerting a negative effect on cytoplasmic maturation of the in vitro oocyte (rabbit: Chung et al. 1974; sow: Yang et al., 2000 and Hong and Lee,

2007). In addition, several authors have suggested that amino acids are among the most important elements involved in regulation of embryonic development, since they are used as energy source and for protein synthesis (woman: Gardner and Lane, 1998 and cow: Lee et al., 2004).

4.2. Non-esterified fatty acids (NEFA)

Ovarian activity is optimized when the animal has sufficient energy reserves primarily of adipose tissue, and has adequate body condition which favors the maximization of reproductive performance in rabbit. In this regard, increased levels of some blood parameters, such as NEFA, reflects the mobilization of lipids from the animal, so it could be a good indicator of energy balance (Walters et al., 2002, Leroy et al., 2005 and Brecchia et al., 2006) observed: a) at the end of gestation, due to the

CHAPTER 1 energy requirements of both the uterus and fetus in growth and reducing feed intake in the days before birth, and b) when pregnancy and lactation overlap (Fortun-Lamothe,

1994). In periods of prolonged fasting, NEFA may increase even three times above normal values in the rabbit (Brecchia et al. 2006). Elevated levels of NEFA induce pro- apoptotic effect on cells granulosa (Vanholder et al., 2005), and have also been associated with decreased quality of bovine in vitro oocytes (Leroy et al., 2005), therefore, it could be considered good indicators of the effect it has on the energy balance effect on the ovarian physiology (Walters et al., 2002).

4.3. Glucose

Glucose may be a good indicator of the energy balance of animals (Chilliard et al., 1998). Long term underfeeding was found to reduce glucose concentration in rabbits

(Rommers et al., 2004), but blood concentration of glucose rises immediately after re- feeding in does fasted from 24-48h (Brecchia et al., 2006). Several authors have observed a fall in glycaemia during the course of gestation in response to the gradual increase in the requirements for foetal growth (Gilbert et al., 1984 and Parigi-Bini,

1988). Blood glucose levels are lower in rabbits which are simultaneously gestating and lactating (Fortun, 1994).

CHAPTER 1

5. Objectives

The general aim of this Doctoral Thesis is to study the effect of different biostimulation strategies on estimated body composition, serum metabolic and ovarian parameters in primiparous lactating rabbits during the post-partum period.

The specific objectives corresponding to the two experimental chapters included in this dissertation:

1- In the First experiment, a study was conducted to assess the influence of early

weaning at 25 dpp on estimated body composition, serum metabolic and ovarian

parameters of primiparous lactating does reared in an extensive reproductive

rhythm (32 dpp).

2- In the second experiment, an extensive rhythm of insemination at 25 dpp was

chosen, eluding early weaning, to compare the most usual management of

insemination at 11 dpp and their effect on live body weight, estimated body

composition, serum metabolic, endocrine parameters and ovarian characteristics

of primiparous lactating does.

CHAPTER 1

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CHAPTER 2

BODY RESERVES AND OVARIAN PERFORMANCE IN

PRIMIPAROUS LACTATING RABBIT DOES

SUBMITTED TO EARLY WEANING AS A STRATEGY

TO DECREASE ENERGY DEFICIT

(EXPERIMENT 1)

Published in: Animal Reproduction Science 121 (2010) 294 – 300

This research was supported by the CICYT project (AGL08-022283) and

the Community of Madrid (S2009/AGR1704).

CHAPTER 2

1. Introduction

In commercial rabbit farms, litter weaning usually occurs from Days 28 to

35 of age. However, primiparous does cannot completely meet the high nutritional demands of lactation and usually show an energy deficit (Cervera et al., 1993; Fortun-

Lamothe, 1998; Parigi Bini and Xiccato, 1998; Xiccato et al., 2004) which reduces their body fat depots and is partly responsible for their low reproductive performance

(Castellini et al., 2006). It has been reported that an extensive reproductive rhythm can be adopted in these animals to improve their nutritional status, fertility, length of reproductive activity and welfare (Castellini et al., 2003; Dal Bosco et al., 2003; Feugier and Fortun-Lamothe, 2006).

In a recent work, it has been shown that insemination of primiparous does at 32 Days post-partum (dpp), only 4 Days after weaning, improved estimated body composition and energy content, serum leptin and protein concentrations, health of follicular populations in the ovary, oocyte quality and, as a consequence, conception rate and prolificacy compared to lactating does inseminated at 11 dpp (Arias-Álvarez et al., 2007). Also, in primiparous does, early weaning between 21 and 25 dpp seems to be a feasible practice to improve their poor body condition before artificial insemination time. Thus, Feugier and Fortun-Lamothe (2006) observed that early weaning at 23 dpp significantly reduced dissectible fat mobilisation of does compared to weaning at 35 dpp. Nevertheless, in both cases, reproductive performance is not affected when females are inseminated at 25 dpp. In turn, Xiccato et al. (2005) have shown that early-weaned does (at 21 or 25 dpp) display a substantial decrease in food intake in the post-weaning period, reducing the daily energy surplus and delaying the complete restoration of their

CHAPTER 2 reserves. In these previous studies, corpora lutea and foetuses were counted, and, when does were inseminated at 25 dpp, the number of corpora lutea and embryonic or fetal mortality were not affected by the age of weaning.

To our knowledge, no studies have been done to assess the consequences of early weaning on ovarian follicle and oocyte features in extensively-reared primiparous rabbit does. In this sense, it is well known that a good ovarian status leads to reproductive success. Additionally, in previous works, high prolactin (PRL) concentrations were observed during lactation in rabbit does (Ubilla et al., 2000). This hormone is the main responsible for the negative effect of lactation on reproductive function (McNeilly et al., 1982). Kermabon et al. (1994) indicated that receptive behavior is correlated with significantly more follicles higher than 1mm of diameter and an increase in the concentration of PRL receptors on the rabbit ovary. Early weaning could decrease the possible inhibitory action of this hormone in follicular development and oocyte quality as described in several species (rabbit: Yoshimura et al., 1989; bovine: Torner et al., 2001).

On the basis of the findings stated above, this study was conducted to assess the influence of early weaning at 25 dpp on estimated body composition, serum metabolic and ovarian parameters of primiparous lactating does when they are reared in an extensive reproductive rhythm.

CHAPTER 2

2. Materials and methods

2.1. Animals

A total of 34 primiparous lactating Californian×New Zealand White rabbit does were used for this study. Animals were housed at the experimental farm in the

Animal Production Department, Polytechnic University of Madrid (Spain), in individual metal cages (50cm×70cm×32 cm) with a closeable nest box, under a constant photoperiod of 16 h light per Day, a temperature of 18–22 ◦C, and a relative humidity of

60–75% maintained by a forced ventilation system. All females were fed ad libitum a commercial diet (NANTA S.A., Tres Cantos, Madrid, Spain) containing 16% crude protein, 15.7% crude fiber and 2.5% fat; digestible energy of the diet was 3900 kcal/kg.

Animals had free access to water. All experimental procedures used in this research were approved by the Animal Ethics Committee of the Polytechnic University of

Madrid and were in compliance with the Spanish guidelines for care and use of animals in research (BOE, 2005).

2.2. Experimental design

Primiparous lactating does were equalized at eight pups one day after parturition, keeping this number constant throughout lactation and dead pups being replaced with others of a similar weight and age provided from nurse does. Thirty-four lactating does were randomly distributed in three experimental groups: 10 lactating does were euthanized on Day 25 pp (group L25), 13 non-lactating does were weaned on Day

25 pp and euthanized on Day 32 pp (group NL32), and 11 lactating does were

CHAPTER 2 euthanized on Day 32 pp (group L32). Live body weight (LBW), estimated body composition and serum metabolic parameters (glucose, non-esterified fatty acids

(NEFA) and total protein) were measured before euthanasia in all groups. Also, solid feed intake of does from parturition until 25 dpp, as well as solid feed intake of does and their litters between 25 and 32 dpp were determined. The solid feed intake of young rabbits from parturition to Day 25 was considered null.

Nevertheless, feed intake of L32 does from Day 25 pp to 32 pp was calculated considering that kits, apart from milk, fed a mean of 30 g of solid feed from Day 25 to

32 of age according to Gidenne and Lebas (2006). Euthanasia was performed with 30 mg/kg of intravenous pentobarbital sodium (Dolethal, Vetoquinol, Alcobendas, Spain).

To further determine ovarian features, both ovaries were recovered by laparotomy after euthanasia. One of them was preserved for histological and immunohistochemical studies (follicular categorization and immunolocalization of prolactin receptor), and the other one was used for in vitro maturation assessment.

2.3. Analytical methods

Unless otherwise stated, all chemicals were purchased from Sigma

Chemical Company (St. Louis, MO, USA).

2.3.1. Body composition

Body composition was estimated by means of bioelectrical impedance analysis (BIA) using a four-terminal body composition analyser (Model Quantum II,

CHAPTER 2

RJL Systems, Detroit, MI, USA) which reports reactance and resistance between two sets of two electrodes. In our case, we used standard 21-gauge needles (Terumo Europe

N.V. 3001 Leuven, Belgium) as electrodes inserted into the skin of the animal. One pair of needles was held 1 cm apart in the neck and the other one in the rump. These components have been correlated to BIA measurements through regression equations built on multiple measurements of rabbit does in different physiological status

(pregnant, lactating, non pregnant, non lactating) and validated according to Nicodemus et al. (2009) and Pereda (2010).

2.3.2. Serum parameters

Blood samples were collected from the margin ear vein into non-heparinized tubes at 9:00 h a.m. to avoid circadian variations and considering that over 60% of the solid feed is consumed by the rabbit in the dark period (Gidenne and Lebas, 2006).

Serum was obtained after centrifugation at 1200×g for 10 min at 4 ◦C and stored at −32

◦C until analysed. Serum non-esterified fatty acids (NEFA) determinations were performed in duplicate samples using a two-reaction enzymatic-based colorimetric assay from Wako Chemicals GmbH Specialty Chemicals (Neuss, Germany). The method is linear over the range from 0.01 to 4.0 mEq/l. Serum glucose was determined in duplicate samples using the GOD-PAP method from RANDOX Laboratories Ltd.

(Crumlin, United Kingdom), according to Tietz (1995). This method is linear up to a glucose concentration of 400 mg/dl. Serum total protein was determined by the Biuret method from RANDOX Laboratories Ltd. (Crumlin, United Kingdom), according to

Tietz (1995). This method is linear up to 13 g/dl.

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2.3.3. Ovarian parameters

Ovary weight, number of follicles ≥1mm on the ovarian surface and hemorrhagic follicles were first recorded. Then, one ovary from each animal was placed into a 4%- buffered neutral paraformaldehyde solution (pH 7.2–7.4). All samples were gradually dehydrated with increasing concentrations of ethyl alcohol, embedded in paraffin, prepared by sectioning at 5μm, and stained with hematoxylin/ eosin. In order to study follicle population, a pair of histological sections of each half ovary was examined at light microscope (Olympus BX40, Hamburg, Germany). Rabbit ovarian follicles were categorized into three specific development stages related to the number of layers of granulosa cells according to Rebollar et al. (2008), namely, primary, preantral and antral follicles. The remaining ovaries were placed in PBS at 37 ◦C.

Cumulus-oocyte complexes (COCs) were obtained from ovarian follicles more than

1mm diameter by aspiration with a 25-gauge needle under a stereoscopic microscope. A total of 317 cumulus-oocyte complexes (group L25, n = 116; group NL32, n = 126; group L32, n = 75) with compact cumulus cells were washed and placed in 500 µl of maturation medium and cultured for 16 h at 38 ◦C under an atmosphere of 5% CO2 in air with maximum humidity. The maturation medium was prepared according to

Lorenzo et al. (1997). After the maturation period, COCs were processed for the confocal study as previously reported by Arias-Álvarez et al. (2007). Nuclear maturation was measured in terms of metaphase II rate. Cortical granule distribution was classified as follows: migrated (CGs were adjacent to the plasma membrane, showing they were cytoplasmically matured) or partially migrated (CGs were spread throughout the cortical area showing they had initiated their cytoplasmic maturation), non-migrated (CGs were distributed throughout the cytoplasm, as they did not show

CHAPTER 2 cytoplasmic maturation) and abnormally migrated (anomalous distribution of CGs compatible with poor quality or degenerated oocytes). To determine PRL receptor on ovarian surface a protocol was used according to that described by Arias-Álvarez et al.

(2009). The primary antibody against prolactin receptor (1:75, Affinity Bioreagents,

Golden, CO, USA) used in this study specifically recognized long and short forms of rabbit prolactin receptor (PRL-R). After detection with diaminobenzidine, sections were counterstained with hematoxylin and photographed under the light microscope

(Olympus BX40). A subjective image analysis was performed to estimate the immunoreactivity of PRL-R in different cell types by an observer unaware of which group of animals was examining. At least five fields of each ovary sample were examined at 400× magnification to evaluate PRL-R immunostaining. Each cell compartment was given an immunohistochemical score, based on the staining intensity

(0–3: 0 = negative, 1 = weak, 2 = intermediate, 3 = strong) according to Picazo et al.

(2004) and García- Palencia et al. (2007).

2.4. Statistical analysis

Trial data were analysed using the Statistical Analysis Systems (SAS/STAT,

1999–2001). Mean differences of LBW, estimated body composition, serum metabolic parameters, feed intake, ovary weight, number of haemorrhagic follicles, follicles more than 1mm diameter, follicular categorization and staining intensity of PRL-R were analyzed with a GLM procedure by a one-way analysis of variance (three differentiated groups L25, NL32 and L32, representing two post-partum times: 25 and 32 dpp). Means were compared using a protected Student’s t-test. In order to compare nuclear maturation and CG migration index of in vitro-matured oocytes among experimental

CHAPTER 2 groups a Chisquare test (proc CATMOD) was carried out. All the results are expressed as mean±S.E.M., and statistical significance was accepted for P < 0.05.

CHAPTER 2

3. Results

3.1. Live body weight, estimated body composition, feed intake and serum

metabolic parameters

Early weaning did not affect LBW of does, which was in average 3.82±1.15 kg. In group NL32, the estimated water content was lower (P < 0.001), and estimated lipids (P < 0.01), protein (P < 0.001), and energy (P < 0.01) body contents were higher than in the two other experimental groups (Table 1). Feed intake from parturition to 25 dpp was similar in all groups, but just as we expected, group L32 had a higher feed intake from 25 to 32 dpp compared to group NL32 (P < 0.0001). Serum NEFA and total protein concentrations did not show significant differences between experimental groups, but serum glucose concentration was lower in group NL32 than in group L32 (P

< 0.04), being intermediate in group L25.

CHAPTER 2

Table 1. Live body weight, estimated body composition, feed intake and serum parameters in primiparous lactating does on Day 25 pp (L25), on Day 32 pp (L32) and in early-weaned, non-lactating does on Day 32 pp (NL32).

Day 25 pp Day 32 pp L25 NL32 L32

No. of does 10 13 11

LBW (kg) 3.86±0.14 3.91±0.94 3.68±1.13

Water (%) 65.9±1.06A 61.5±0.89B 66.9±1.07A

Ash (%) 3.09±0.05 3.04±0.04 3.14±0.04

Lipids (%) 13.4±1.03b 16.9± 0.09a 11.9±1.31b

Proteins (%) 18.5±0.10B 19.7±0.07A 18.5±0.08B

Energy (MJ/kg) 993±40.4b 1147±2.7a 942±51.3b

Feed intake (g/Day)

Parturition to 25 Days pp 345±19.6 357±17.2 359±17.9

25 to 32 Days pp1 - 122±23.5A 402±26.7B

Serum concentrations

NEFA (mmol/L) 0.26±0.04 0.23± 0.03 0.19±0.04

Glucose (mg/dl) 212±27.9 ab 158±24.5 b 259±29.5 a

Total protein (g/dl) 7.60±1.26 7.40±1.10 6.50±1.33

pp: post-partum. LBW: Live Body Weight. NEFA: Non-esterified fatty acids.

A, B: P < 0.001; a, b: P < 0.01 in the same row.

1Calculated considering litter size of each doe (8 kits) and a mean dry feed intake of kits of 30 g/d.

CHAPTER 2

3.2. Features of ovarian follicular population

As shown in Table 2, no significant differences were found in average ovary weight in the number of haemorrhagic follicles between experimental groups.

Nevertheless, L25 does showed a lower number of follicles ≥1mm in the ovarian surface per ovary than groups NL32 and L32 (P < 0.05). Follicular population observed in the histological sections was not affected by early weaning, and similar means of primary, preantral and antral follicles in the ovarian sections were found (11.4±1.71,

13.7±1.84, and 13.3±1.12 follicles per ovary, respectively; P>0.05). PRL-R cytoplasmic staining was observed in all ovarian compartments studied (granulose cells of all categorized follicles and also in corpora lutea (CL) and stromal cells), but not in theca cells, as shown in Fig. 1. No staining was detected in the negative controls. No statistical differences were found in the staining intensity of PRL-R in different size follicles and CL in group L25 compared to groups L32 and NL32.

3.3. In vitro oocyte maturation

As shown in Table 2, Metaphase II rate observed in oocytes from non- lactating does was significantly higher (P < 0.05) than in oocytes from lactating does at

25 dpp (L25), but similar to lactating rabbits at 32 dpp. Percentage of cytoplasmic maturation was significantly different between groups (P < 0.05). In group NL32, a significantly higher rate of oocytes with migrated or partially migrated CGs was found with respect to L25 (P < 0.05), but similar to L32 (P = 0.06). Oocytes with no migrated

CGs were higher for group L25 than for the two other groups (P < 0.05). No significant differences were found among groups regarding abnormal migration of CGs.

CHAPTER 2

Table 2. Ovarian status in primiparous lactating does and in vitro oocyte maturation from primiparous lactating does on Day 25 pp (L25), on Day 32 pp (L32), and in early- weaned, non-lactating does on Day 32 pp (NL32). Nuclear and cytoplasmic maturation were measured in terms of metaphase II rate (MII) and cortical granule (CG) migration.

Day 25 pp Day 32 pp L25 NL32 L32

No. of does 10 13 11

Ovary Weight (mg)

Right 347±37.4 329±34.1 295±39.4

Left 331±32.9 323±30.1 286±34.7

Follicles (n)

>1mm 12.7±1.58b 18.0±1.45a 17.6±1.67a

Haemorrhagic 1.30±0.85 1.00±0.77 0.90±0.89

Mean of follicular population/ovary (n)

Antral 9.88±2.17 15.7±2.05 13.3±1.70

Preantral 12.9±3.6 19.3±3.36 10.4±2.80

Primary 10.8±3.32 13.4±3.13 10.4±2.61

In vitro oocyte maturation

MII rate (%) 67.0 b 79.7 a 78.3 ab

Migrated or partially migrated 16.0 b 38.3 a 60.0 a CGs (%) Non-migrated CGs (%) 76.0 a 46.8 b 33.3 b

Abnormally migrated CGs (%) 8.0 14.9 3.3 pp: post-partum. a,b: P < 0.05 in the same work.

CHAPTER 2

Figure 1. Immunohistochemical localization of PRL-R in ovaries of does early weaned at 25 days post-partum (group L25 and group NL 32) or non-weaned (group L32) in the different sized follicles and corpus luteum. (A) Negative control (20×); (B) primary follicles (PF) (40×) with positive (brown) granulose cells and oocytes, surrounded by positive stromal cells (SC); (C) antral follicle (AF) showing strong immunoreactivity to

PRL-R in granulosa cells, and negative theca cells (20×); (D) Corpus luteum (CL) (20×) showing positive immunostaining in all luteal cells. Given that the pattern and intensity of localizations did not change between ovaries of the three experimental groups, only representative images of follicles and corpus luteum are shown (A–D). Scale bar: 100

µm.

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4. Discussion

Early weaning is one factor to settle an extensive reproductive rhythm. From the ovarian and energy points of view, the present study will clarify some aspects of this critical reproductive period.

4.1. Live body weight, estimated body composition, feed intake and serum

metabolic parameters

LBW of does on Day 25 pp was similar to that observed in primiparous does in previous studies (Rebollar et al., 2009). The greatest interest in early weaning techniques lies in the possibility of reducing doe body energy deficit. In this sense, estimated body composition and energy changes observed in this work have confirmed previous results found on primiparous lactating rabbit does (Xiccato et al., 2005;

Fortun-Lamothe and Prunier, 1999; Feugier and Fortun-Lamothe, 2006; Castellini et al.,

2006). A high energy supply is required during the final lactation period, since milk dry matter and lipid concentrations increase from Day 20 onwards when production begins falling (Lebas, 1971, 1972; Pascual et al., 1999). Using ultrasounds, Pascual et al.

(2002) observed a decrease of around 0.2mm in peri-renal fat thickness during the first three weeks of lactation, rising to a loss of 0.9mm in the last week. Similar results were obtained by Fortun-Lamothe et al. (2002), who found a decrease of 5MJ in the total energy content of primiparous does during lactation, or Fortun-Lamothe and Prunier

(1999), who reported a weight loss (−97 g per week) after the first two weeks post- partum. In our study, focused on the last week of lactation, primiparous lactating does at

32 dpp maintained similar body energy reserves than lactating does at 25 dpp.

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Nevertheless, weaning at 25 dpp increased energy reserves; non-lactating animals had 154 and 205 MJ/kg more body energy content than lactating does at 25 and

32 dpp, respectively. Although feed intake was lower in weaned does, decreased milk production for seven days was enough to improve slightly their energy reserves.

Consequently, body lipids and protein content were also improved in non-lactating does. Previous studies observed that weaning age of litters strongly influence the evolution of the body condition of females during their reproductive cycles. Early weaning (23 Days of age, Feugier and Fortun-Lamothe, 2006; 21 Days of age, Xiccato et al., 2005) reduces fat loss and energy deficit with respect to weaning performed later

(33 Days of age, Feugier and Fortun-Lamothe, 2006, or 25 Days of age, Xiccato et al.,

2005, respectively). This improvement on body condition is observed even though the feed intake of does is voluntarily reduced, according to our results. This is due to the chemostatic mechanism of appetite regulation according to which, after litter weaning, does quickly decrease their feed intake by 35–45% of the lactation level in just a week

(Xiccato et al., 2005).

The lower serum glucose concentrations observed in non-lactating does on Day

32 pp compared to lactating ones could be explained by the substantial decrease in feed intake during the dry period after weaning, reducing the daily energy intake as Xiccato et al. (2004) demonstrated. When serum glucose concentrations fall, NEFA must be released into the blood stream (Brecchia et al., 2006). In fact, around parturition, elevated concentrations of NEFA have been previously described due to mobilization of body reserves and reduced feed intake at the end of pregnancy (AriasÁlvarez et al.,

2009). Nevertheless, neither lactating does at 25 dpp nor weaned ones at 32 dpp showed differences in NEFA concentrations. In that sense, Arias-Álvarez et al. (2009)

CHAPTER 2 did not observe differences among does on Day 11 pp and on Day 32 pp. As regards both serum parameters, low concentrations of glucose are not enough to evoke any meaningful effect on the evolution of NEFA concentrations in lactating rabbit does. In fact, Xiccato et al. (2005) observed a high individual variability in NEFA concentrations of does under different reproductive rhythms and weaning ages.

4.2. Features of ovarian follicular population

Ovarian features were similar in lactating and nonlactating does at 32 dpp.

Only the post-partum times considered (25 or 32 dpp) had a significant effect in mean preovulatory follicles, since they were lower on Day 25 pp than on Day 32 pp. The reduction in the number of preovulatory follicles observed in group L25 is consistent to their lower estimated body composition, which corresponds to the negative energy balance and the endocrine changes expected in these animals, changes affecting in turn follicular growth (Diskin et al., 2003).

The distribution or evolution of follicular waves in the rabbit ovary is not well described. These animals continuously produce mature follicles which become atretic if ovulation is not stimulated by coitus (Kranzfelder et al., 1984). Ubilla and Rebollar

(1995) suggested the presence of a follicular growth wave between Days 23 and 30 of the post-partum period in non-pregnant lactating rabbits. This wave coincided with an increase in plasma oestradiol levels found on Days 23–30 after parturition and with a rise of sexual receptivity. Current results show the existence of this physiological follicular wave irrespective of whether rabbit does were lactating or not. Glucose, insulin and leptin are the most likely signals informing the hypothalamus of the animal

CHAPTER 2 metabolic status (Diskin et al., 2003). In our work, follicular growth took place irrespective of glucose concentrations availability because lactating and non-lactating does at 32 dpp had different serum glucose concentrations.

Prolactin effects are related to the presence of its receptor. Prolactin concentration fluctuates according to lactation stage (Durand and Djiane, 1977), and it acts directly on rabbit follicles, modulating follicular health and development (Ben-

Jonathan et al., 1996). In this study, PRL-R appeared in different ovarian localizations, including granulosa cells and antral follicle oocytes, matching previous results

(Kermabon et al., 1994; García-García et al., 2007). However, we found no significant differences in the cellular localization of prolactin receptor among groups. Matching our results, previous studies showed no differences between lactating and non-lactating rabbit does (Kermabon et al., 1994; Arias-Álvarez et al., 2009). Some works have shown that localization of PRL-R exhibits remarkable changes throughout the oestrus cycle in ewe (Picazo et al., 2004), which is closely regulated by hormones and/or local factors. In our work, rabbits are probably having the physiological follicular wave on

Days 23–30, pp, as is suggested by Ubilla and Rebollar (1995), so time of oestrus cycle and endocrine environment, including distribution of PRL receptor could be similar for all groups.

4.3. In vitro oocyte maturation

Oocyte maturation implies both nuclear and cytoplasmic maturation including CG migration (Edwards, 1965; Yanagimachi, 1994). A recent report in rabbit

(AriasÁlvarez et al., 2009) clearly shows that conditions related to early lactation and

CHAPTER 2 negative energy balance adversely affect oocyte quality and endocrine parameters, effects which are especially pronounced after the first parturition. In the current study, on Day 32 pp oocytes matured in vitro from ovaries of lactating or non-lactating females showed higher nuclear and cytoplasmic maturation rates than oocytes of lactating does on Day 25 pp. Additionally, the percentage of cytoplasmic immature oocytes, with non-migrated CGs, was also higher in does on Day 25 of lactation. These results confirm the previously described progressive improvement of reproductive performance during lactation (Ubilla and Rebollar, 1995; Feugier and Fortun-Lamothe,

2006).

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5. References

Arias-Álvarez, M., López-Béjar, M., Rebollar, P. G., García-García, R. M. & Lorenzo, P.L., 2007. Nuclear and cytoplasmic patterns of rabbit IVM oocytes. Investigación Técnica Económica Agraria 28, 42–44. Arias-Álvarez, M., García-García, R. M., Rebollar, P. G., Revuelta, L., Millán, P. & Lorenzo, P.L., 2009. Influence of metabolic status on oocyte quality and follicular characteristics at different post-partum periods in primiparous rabbit does. Theriogenol., 72, 612–623. Ben-Jonathan, N., Mershon, J. L., Allen, D. L. & Steinmetz, R. W., 1996. Extrapituitary prolactin: distribution, regulation, functions, clinical aspects. Endocr. Rev., 17, 639–669. BOE. Boletín Oficial del Estado, 2005. Real Decreto 1201/2005. Sobre protección de los animales utilizados para experimentación y otros fines científicos. BOE, 252, 34367–34391. Brecchia, G., Bonanno, A., Galeati, G., Federici, G., Maranesi, M., Gobbetti, A., Zerani, M. & Boiti, C., 2006. Hormonal and metabolic adaptation to fasting: effects on the hipotalamic–pituitary–ovarian axis and reproductive performance of rabbit does. Domest. Anim. Endocrin,. 31, 105–122. Castellini, C., Dal Bosco, A. & Mugnai, C., 2003. Comparison of different reproductive protocols for rabbit doe: effect of litter size and remating interval. Livest. Prod. Sci., 83, 131–139. Castellini, C., Dal Bosco, A. & Cardinali, C., 2006. Long term effect of postweaning rhythm on the body fat and performance of rabbit doe. Reprod. Nutr. Dev., 46, 195–204. Cervera, C. J., Fernández-Carmona, J., Viudes, P. & Blas, E., 1993. Effect of remating interval and diet on the performance of female rabbits and their litters. Anim. Prod., 56, 399–405. Dal Bosco, A., Castellini, C. & Cardinali, R., 2003. Evaluation of body condition score in pregnant rabbit does by ultrasound scanner. In Proceeding of the Atti XV Congresso. ASPA, Parma, pp. 480–482.

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Diskin, M. G., Mackey, D. R., Roche, J. F. & Sreenan, J. M., 2003. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Anim. Reprod. Sci., 78, 345–370. Durand, P. & Djiane, J., 1977. Lactogenic activity in the serum of rabbits during pregnancy and lactation. Endocrinology, 75, 33–42. Edwards, R. G., 1965. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208, 349–351. Feugier, A. & Fortun-Lamothe, L., 2006. Extensive reproductive rhythm and early weaning improve body condition and fertility of rabbit does. Anim. Res., 55, 459–470. Fortun-Lamothe, L., 1998. Effects of pre-mating energy intake on reproductive performance of rabbit does. Anim. Sci., 66, 263–269. Fortun-Lamothe, L., Prunier, A., 1999. Effects of lactation, energetic deficit and remating interval on reproductive performance of primiparous rabbit does. Anim. Rep. Sci., 55, 289–298. Fortun-Lamothe, L., Lamboley-Gaüzère, B., Bannelier, C., 2002. Prediction of body composition in rabbit female using total body electrical conductivity (TOBEC). Livest. Prod. Sci. 78, 132–142. García-García, R. M., Arias-Álvarez, M., García-Palencia, P., Revuelta, L., Sánchez- Maldonado, B., Rebollar, P. G. & Lorenzo, P. L., 2007. Localización del receptor de prolactina en el ovario de conejas en diferentes estados fisiológicos. II Congreso Ibérico de Cunicultura, In el acta de IV Jornadas Internacionales de Cunicultura y XXXII Symposium de Cunicultura. 5,6 junio. Ed. Asociación Española de Cunicultura, Vila Real (Portugal), 41–46. García-Palencia, P., Sánchez, M. A., Nieto, A., Vilar, M. P., González, M., Veiga- López, A., González-Bulnes, A. & Flores, J. M., 2007. Sex steroid receptor expression in the oviduct and uterus of sheep with estrus synchronized with progestagen or prostaglandin analogues. Anim. Reprod. Sci., 97, 25–35. Gidenne, T. & Lebas, F., 2006. Feeding behaviour in rabbits. In: Bels, V. (Ed.), Feeding in Domestic Vertebrates. From Structure to Behaviour. CABI Publishing, Wallingford, UK, 179–209, (Chapter 11). Kermabon, A. Y., Belair, L., Theau-Clement, M., Salesse, R. & Djiane, J., 1994. Effects of anoestrus and bromocryptine treatment on the expression of prolactin and LH receptors in the rabbit ovary during lactation. J. Reprod. Fertil., 102, 131–138. 94

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Kranzfelder, D., Korr, H., Mestwerdt, W. & Maurer-Schultze, B., 1984. Follicle growth in the ovary of the rabbit after ovulation-inducing application of human gonadotrophin. Cell Tissue Res., 238, 611–620. Lebas, F., 1971. Composition chimique du lait de lapine, évolution au cours de la traite et en fonction du stade de lactation. Ann. Zootech., 20, 185–191. Lebas, F., 1972. Effet de la simultanéité de la lactation et de la gestation sur les performances latières chez la lapine. Ann. Zootech., 21, 129–191. Lorenzo, P. L., Illera, J. C., Silván, G., Munro, C. J., Illera, M. J. & Illera, M., 1997. Steroid-level response to insulin-like growth factor-1 in oocytes matured in vitro. J. Reprod. Immunol., 35, 11–29. McNeilly, A. S., Glasier, A., Jonassen, J. & Howie, P. W., 1982. Evidence for direct inhibition of ovarian function by prolactin. J. Reprod. Fertil., 65, 559–569. Nicodemus, N., Pereda, N., Romero, C. & Rebollar, P. G., 2009. Évaluation de la technique d’impedance bioélectrique (IBE) pour estimer la composition corporelle de lapines reproductrices. In: Bolet, G. (Ed.), 13 èmes Journées de la Recherche Cunicole. LeMans, France, 109–112. Pascual, J. J., Cervera, C., Blas, E. & Fernández-Carmona, J., 1999. Effect of high fat diets on the performance, milk yield and milk composition of multiparous rabbit does. Anim. Sci., 68, 151–162. Pascual, J. J., Motta, W., Cervera, C., Quevedo, F., Blas, E. & Fernández- Carmona, J., 2002. Effect of dietary energy source on the performance and peri-renal fat thickness evolution of primiparous rabbit does. Anim. Sci., 75, 267–279. Parigi Bini, R. & Xiccato, G., 1998. Energy metabolism and requirements. In: De Blas, J.C., Wiseman, J. (Eds.), The Nutrition of the Rabbit. CABI Publishing, Wallingford, UK, 103–131. Pereda, N., 2010. Evaluación de la técnica del Análisis de Impedancia Bioeléctrica para predecir la composición corporal: aplicación en conejas sometidas a diferentes sistemas de alimentación durante la recría. In: Tesis Doctoral. Universidad Politécnica de Madrid, Madrid, Spain, 194. Picazo, R. A., García Ruiz, J. P., Santiago Moreno, J., González de Bulnes, A., Mu˜noz, J., Silván, G., Lorenzo, P. L. & Illera, J. C., 2004. Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle. Reprod., 128, 545–553.

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Rebollar, P. G., Bonanno, A., Di Grigoli, A., Tornambè, G. & Lorenzo, P. L., 2008. Endocrine and ovarian response after a 2-Day controlled suckling and eCG treatment in lactating rabbit does. Anim. Reprod. Sci., 104, 316–328. Rebollar, P. G., Pérez-Cabal, M. A., Pereda, M. A., Lorenzo, P. L., Arias-Álvarez, M. & García-Rebollar, P., 2009. Effects of parity order and reproductive management on the efficiency of rabbit productive systems. Livest. Sci., 123, 107–115. SAS/STAT, 1999–2001. SAS 7. In Proceeding of the STAT User’s Guide (Release 8.2). SAS Inst. Inc., Cary, NC. Tietz, N.W., 1995. Clinical Guide to Laboratory Tests, 3rd Ed. W.B. Saunders Co., Philadelphia, 518–519. Torner, H., Kubelka, M., Heleil, B., Tomek, W., Aim, H., Kuzmina, T. & Guiard, V., 2001. Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size. Theriogenol., 55, 885–899. Ubilla, E. & Rebollar, P. G., 1995. Influence of the postpartum day on plasma oestradiol-17_ levels, sexual behavior and fertility rate in artificially inseminated lactating rabbits. Anim. Reprod. Sci., 38, 337–344. Ubilla, E., Rebollar, P. G., Pazo, D., Esquifino, A. & Alvariño, J. M., 2000. Effects of doe litter separation on endocrinological and productivity variables in lactating rabbits. Livest. Sci., 67, 67–74. Xiccato, G., Trocino, A., Sartori, A. & Queaque, P. I., 2004. Effect of parity order and litter weaning age on the performance and body energy balance of rabbit does. Livest. Prod. Sci., 16, 239–251. Xiccato, G., Trocino, A., Boiti, C. & Brecchia, G., 2005. Reproductive rhythm and litter weaning age as they affect rabbit doe performance and body energy balance. Anim. Sci., 81, 289–296. Yanagimachi, R., 1994. Mammalian fertilization. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press, 189–279. Yoshimura, Y., Hosoi, Y., Irritan, A., Nakamura, Y., Atlas, S. J. & Wallach, E. E., 1989. Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin. Biol. Reprod., 41, 26–33.

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METABOLIC AND REPRODUCTIVE STATUS

ARE NOT IMPROVED FROM 11 TO 25 DAY

POST-PARTUM IN NON WEANED

PRIMIPAROUS RABBIT DOES

(EXPERIMENT 2)

Published in: Animal Reproduction Science 131 (2012) 100 – 106.

This research was supported by the CICYT project (AGL08-022283), the

Community of Madrid (S2009/AGR1704) and the UCM research program

(920249)

CHAPTER 3

1. Introduction

Artificial insemination (AI) during lactation is a common practice in commercial rabbit farms. However, the reproductive outcome of high milk producers, such as cattle or rabbits decreases due to the negative energy balance of females (NEB) around parturition and in early lactation (cows: Jorritsma et al., 2003; rabbits: Parigi-

Bini et al., 1992). The difference between energy uptake and energy requirements for lactation of multiple litters and gestation provokes NEB. This situation is more critical in primiparous rabbit does because females have also energetic needs for their own growth (Xicatto et al., 2005).

The AI technique requires the use of more or less intensive fixed reproductive rhythms. Long-term reproductive performance of the doe is negatively affected and litter viability decreases using intensive reproductive management (AI on Day 4 pp) compared to semi-intensive (AI on Day 11 pp) (Rebollar et al., 1994; 2009; Garcia-

Garcia et al., 2009). Thus, 11 days post-partum (dpp) insemination time corresponds to one of the most used reproductive rhythms. However, in recent years a more

“physiologically adapted” extensive and post-weaning rhythm has been proposed to improve reproduction performance in primiparous does (Castellini et al., 2003).

Previous studies of our group have demonstrated better body condition and metabolic status of primiparous rabbit does inseminated at 32dpp and weaned four days before compared to rabbit does at 11 dpp. In these earlier inseminated females, ovarian follicle, oocyte quality and reproductive performance (conception rate and prolificacy) are negatively affected (Arias-Álvarez et al., 2009). An early weaning

CHAPTER 3 strategy involves adapting post-weaning feeding of kits because their digestive tract is not quite ready. Although feed intake of rabbits weaned at 25 days is adequate, their liveweight at 35 days old is lower than animals weaned later (at 35 dpp) and their mortality is higher (Rebollar et al., 2009). In the first experiment we have demonstrated that early weaning at 25 dpp improves the energy status of the primiparous rabbit does inseminated at 32 dpp, but ovarian parameters were similar to those of non-weaned rabbits and consequently it is recommended to avoid early weaning. On the other hand, with an extensive rhythm, the interval from parturition to insemination time usually increases. If rabbit does lose their litters during this time ( very common in primiparous rabbits) there would be a serious risk of fatness, which impairs fertility (Castellini et al.,

2006).

Based on these findings we decided to examine an alternative extensive rhythm of insemination at 25 dpp instead of 32 dpp, to decrease the parturition-insemination interval, avoiding early weaning to improve kit viability and compare it to the usual 11 dpp insemination. To assess body and ovarian status of these females before insemination, we evaluated the metabolic (live weight, estimated body composition, serum non- esterified fatty acids (NEFA), glucose and protein concentrations) and endocrine status (serum steroids concentrations), and the ovarian characteristics

(follicular population categorization, follicular atresia, and oocyte maturation) of primiparous lactating does submitted to the semi-intensive and extensive reproductive rhythms described above.

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2. Materials and methods

Unless otherwise stated, all chemicals were purchased from Sigma Chemical

Company (St. Louis, MO, USA). All the experimental procedures were approved by the

Animal Ethics Committee of the Polytechnic University of Madrid (Spain), in compliance with the Spanish guidelines for the care and use of animals in research

(BOE, 2005).

2.1. Animals and experimental design

A total of 48 primiparous lactating Californian x New Zealand White rabbit does were housed at the experimental farm in the Animal Production Department,

Polytechnic University of Madrid (Spain), in individual flat-deck metal cages (700 mm x 500 mm x 320 mm) with a closeable nest box, under a constant photoperiod of 16 h light per day.A temperature of 18–22°C, and a relative humidity of 60–75% was maintained by a forced ventilation system. All females were fed ad libitum a commercial diet (NANTA S.A., Tres Cantos, Madrid, Spain) containing 16.9% crude protein, 15.7% crude fiber and 2.5% fat; digestible energy of the diet was 3900 kcal/kg.

Animals had free access to water.

Pups were reallocated to does from 1 day post parturition to ensure 8 pups per doe throughout lactation. Dead pups were replaced with others of a similar weight and age, provided from nurse does. The rabbit does were randomly allocated in: a) semi- intensive rhythm group (n=24), lactating does euthanized at early post-partum period

(11 dpp) and b) extensive rhythm group (n=24), lactating does euthanized on later post-

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CHAPTER 3 partum period (25 dpp). Live weight (LW), body composition and serum metabolic and endocrine parameters were recorded at parturition time and at 11 and 25 dpp. Ovarian parameters were measured at 11 and 25dpp.

2.2. Analytical methods

2.2.1. Body composition

Body composition was estimated by bioelectrical impedance analysis (BIA) using a four-terminal body composition analyser (Model Quantum II, RJL Systems,

Detroit, MI, USA) which reports reactance and resistance between two sets of two electrodes. We used standard 21-gauge needles (Terumo Europe N.V. 3001 Leuven,

Belgium) as electrodes inserted into the skin of the animal. One pair of needles was held

1 cm apart in the neck and the other one in the rump. These components have been correlated to BIA measurements through regression equations based on multiple measurements of rabbit does in different physiological status and validated according to

Pereda (2010).

2.2.2. Serum metabolic and endocrine parameters

Blood samples were collected from the margin ear vein into non-heparinized tubes at 9:00 h a.m. to avoid circadian variations in feed intake (Gidenne and Lebas,

2006). Serum was obtained after centrifugation at 1200 g for 10 min at 4ºC and stored at

-32ºC until analyzed.

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Serum non-esterified fatty acids (NEFA) determinations were performed in duplicate samples using a two-reaction enzymatic-based colorimetric assay from Wako

Chemicals GmbH Specialty Chemicals (Neuss, Germany). The method is linear over the range from 0.01 to 4.0 mEq L-1.

Serum glucose was determined in duplicate samples using the GOD-PAP method from RANDOX Laboratories Ltd. (Crumlin, United Kingdom), according to

Tietz (1995). This method is linear up to a glucose concentration of 400 mg 100mL-1.

Serum total protein was determined by the Biuret method from RANDOX

Laboratories Ltd. (Crumlin, United Kingdom), according to Tietz (1995). This method is linear up to 13g100mL-1.

Oestradiol-17β (E2) and Progesterone (P4.). Serum E2 and P4 concentrations were measured in duplicate samples by specific chemiluminiscence methods (CMIA, Abbott laboratories, Abbott Park II., USA). For E2 detection, purified rabbit anti-oestradiol monoclonal antibodies were used. The P4 detection was achieved by using rabbit anti- progesterone polyclonal antibodies. Intra and inter-assay coefficients of variation were

6.6% and 7% for E2, and 5.8% and 6.3% for P4, respectively. The detection limit was

-1 -1 10 pg mL (E2) and 0.2 ng mL (P4).

2.2.3. Study of ovary weight and follicular population

Ovary weight, number of follicles ≥1 mm on the ovarian surface were recorded for both ovaries. The left ovary from each animal was then placed into a 4%-

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CHAPTER 3 buffered neutral paraformaldehyde solution (pH 7.2–7.4). All samples were gradually dehydrated with increasing concentrations of ethyl alcohol then embedded in paraffin and prepared for sectioning longitudinally at 5 μm, and stained with hematoxylin/eosin.

In order to study follicle population, four histological sections of the ovary was examined with a light microscope (Olympus BX40, Hamburg, Germany). Rabbit ovarian follicles were categorized into four specific development stages related to the number of layers of granulosa cells according to Rebollar et al. (2008) and Hutt et al.

(2006) as primordial, primary, preantral and antral follicles. Briefly, primordial follicles were surrounded for a single layer of flattened granulose cells; primary follicles had a single layer of cuboidal granulosa cells whereas preantral follicles had 2 to <4 layers of cuboidal granulosa cells; >5 layers of granulosa cells and antrum were observed in antral follicles.

2.2.4. Follicular atresia analysis

Strand breaks of DNA that occur during cell apoptosis were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL; In

Situ Cell Death Detection Kit, POD, Roche Diagnostics S.L., Applied Science,

Barcelona, Spain). Dewaxed, rehydrated sections were prepared using standard methods and the slides were pretreated with 20 μg mL-1 of proteinase K working solution for 30 minutes in a humidified dark chamber at 37°C. Incubation with the TUNEL reaction mixture took place also in a humidified dark chamber at 37°C, for 1 hour. After each step of the procedure, sections were rinsed three times in phosphate-buffered saline

(PBS). Finally, the slides were covered with Vectashield mounting medium with 4´,6- diamino-2-phenylindole (DAPI) (Vector Laboratories, Ltd., Peterborough, UK).

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Positive control sections were treated with DNAse I (Roche Diagnostics S.L., Applied

Science, Barcelona, Spain) for 10 minutes at room temperature in a humidified chamber before incubation with the TUNEL reaction mixture. For negative controls, samples were incubated with the label solution of the TUNEL reaction mixture without the enzymatic solution. TUNEL-stained and DAPI-counterstained slides were observed under a fluorescent microscope (Leica, F550, Wetzlar, Germany). Green fluorescence could be visualized only in TUNEL-positive cells.

A total of 426 antral follicles were examined. Follicles in medium or advanced stage of atresia were recorded. According to Kasuya et al. (1995) and Arias-Álvarez et al. (2010), medium atretic follicles show less than 50% of the granulose cells labelled whereas advanced atretic follicles were identified as those in which apoptosis occur in more than 50% of granulosa cells. Apoptosis was expressed as the number of TUNEL- positive follicles divided by the total number of recorded follicles.

2.2.5. Oocyte collection and in vitro maturation

The remaining right ovaries were placed in PBS at 37°C. Cumulus-oocyte complexes (COCs) were obtained from ovarian follicles larger than 1mm in diameter by aspiration with a 25-gauge needle under a stereoscopic microscope (Nikon, Tokio,

Japan). Cumulus-oocyte complexes (COC) with compact cumulus cells were washed and placed in 500 μl of maturation medium in four-well dishes (Nunclon Surface, Nunc,

Roskilde, Denmark). They were cultured for 16 h at 38°C under an atmosphere of 5%

CO2 in air with maximum humidity. The maturation medium was prepared according to

Garcñia-Garcñia et al. (2009).

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2.2.6. Confocal microscopy study

After the maturation period, a total of 242 COCs were processed for the confocal study as previously reported by Arias-Álvarez et al. (2009). Nuclear maturation was measured in terms of metaphase II rate. Cortical granule (CGs) distribution was classified as migrated (CGs were adjacent to the plasma membrane), showing they were cytoplasmically matured, partially migrated (CGs were spread throughout the cortical area), considered partially matured, homogeneous (CGs were distributed throughout the cytoplasm), cytoplasmically matured and abnormally migrated (anomalous distribution of CGs), which is compatible with poor quality or degenerated oocytes.

2.3. Statistical analysis

Data were analysed using the Statistical Analysis Systems (SAS/STAT 1999-

2001). Mean differences of LW, body composition, serum metabolic parameters from parturition to 11 dpp or until 25 dpp were studied by repeated measures analysis using the MIXED Procedure of SAS (Littell et al., 1996). Mean differences of LW, body composition, serum metabolic parameters, ovary weight, number of follicles more than

1mm of diameter, follicular categorization and follicular atresia rate between the two post-partum times (11 vs. 25 dpp) were analyzed with a GLM procedure by a one-way analysis of variance. Means were compared using a protected t-test. In order to compare nuclear maturation and CG migration index of in vitro-matured oocytes among experimental groups, a Chi-square test (proc CATMOD) was carried out. All the results are expressed as mean ± S.E.M. and statistical significance was accepted at P  0.05.

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3. Results

3.1. Body composition and metabolic status

3.1.1. Live weight (LW) and body composition

Live weight decreased significantly (P < 0.05) between parturition time and the two post-partum periods studied and also between 11 and 25dpp (see Table 1).

Estimated body composition was also significantly different between the three periods measured. Thus, the body lipids depots diminished throughout the post-partum period

(P < 0.0002) whereas the body energy content of does decreased from day 11 to 25 pp

(P < 0.0007). The body protein and ash contents increased significantly throughout the post-partum period (P < 0.03 and P < 0.003, respectively). Water content decreased at

11 dpp and then increased at 25 dpp (P < 0.001).

Table 1. Effect of day post-partum on live weight and estimated body composition of primiparous lactating rabbit does.

Parturition 11 dpp 25 dpp

Nº of does 24 24 24

Live weight (Kg) 4.46 ± 0.06 a 4.13 ± 0.07 b 3.89 ± 0.09 c

Water % 63.9 ± 0.3 b 61.9 ± 0.4 a 64.6 ± 0.6 b

Ash % 2.8 ± 0.0 c 3.0 ± 0.0 b 3.1 ± 0.0 a

Lipids % 19.1 ± 0.3 a 18.3 ± 0.5 b 14.5 ± 0.8 c

Protein % 17.7 ± 0.0 c 18.3 ± 0.0 b 18.4 ± 0.1 a

Energy % (Kj/100g) 1152 ± 23.1 a 1188 ± 20.6 a 1049 ± 31.5 b

Different letters in the same row indicate significant differences (P < 0.05) dpp: days post-partum.

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3.1.2. Metabolic parameters

The highest serum NEFA concentrations were observed at parturition (P <

0.02; Figure 1A). However, there was no difference between day 11 and 25 pp

(P=0.09). On the other hand, glucose concentrations were lower at parturition time than in the other two moments studied (P < 0.002; Figure 2A) but no differences were found between 11 and 25 dpp (P>0.05). The same trends were found for serum protein levels although albumin concentrations did not vary significantly throughout the post-partum period (Figure 1B and 2B, respectively).

Figure 1. (A) Serum non esterified fatty acids (NEFA) and (B) protein concentrations measured at parturition and post-partum (pp) period (11 and 25 days pp) in primiparous lactating rabbit does. Different letters indicate significant differences (P < 0.05).

0,8 (A) 10 (B)

a 8 b 0,6 b

6 a 0,4 b 4 b 0,2

Serum protein (g/dl) Serum protein 2 Serum NEFA (mmol/l)

0 0 Parturition Day 11pp Day 25pp Parturition Day 11pp Day 25pp

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Figure 2. (A) Glucose and (B) albumin concentrations measured at parturition and post- partum (pp) period (11 and 25 days pp) in primiparous lactating rabbit does. Different letters indicate significant differences (P < 0.05).

250 (A) 6 (B) b 200 b 5 4 150 a 3 100 2 50 Serum albumin (g/dl) Serum albumin Serum glucose (mg/dl) Serum glucose 1

0 0 Parturition Day 11pp Day 25pp Parturition Day 11pp Day 25pp

3.2. Ovarian features

3.2.1. Steroid concentrations, ovary weight, follicular population and atresia rate index analysis

Serum oestradiol and progesterone concentrations were similar (P>0.05) at 11 and 25 dpp (41.3 ± 1.8 vs. 40.9 ± 1.7 pg/mL and 1.1 ± 0.3 vs. 2.0 ± 0.7 ng/mL, respectively).

Average ovary weights were similar for the two post-partum groups (300.2 ±

28.9 vs. 310.3 ± 20.9 mg). The average of total preovulatory follicles on the ovarian surface per ovary was significantly higher at 11 dpp than 25 dpp (18.0 ± 1.0 vs. 15.2 ± ever, no statistical differences were found in the rest of the histological classified

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CHAPTER 3 follicular population (mean of primordial follicles: 116.5 ± 10.3 vs. 128.7 ± 16.4; primary follicles: 20.0 ± 2.0 vs. 21.8 ± 3.8; preantral follicles: 12.6 ± 1.7 vs. 13.0 ± 1.6 and antral follicles: 6.4 ± 0.8 vs. 6.2 ± 1.0).

In addition, the atresia rate was slightly higher in 25 dpp than in 11 dpp (28.8 ±

2.4 vs. 23.2 ± 3.1%; P < 0.1) and accordingly, the percentage of healthy follicles also tended to be lower at day 25 pp (49.4 ± 2.9 vs. 56.9 ± 3.2%; P < 0.09). A similar percentage of early atretic follicles were found in both groups (21.8 ± 3.2 vs. 19.9 ±

2.8%).

3.2.2. Nuclear and cytoplasmic oocyte maturation

The rate of oocytes showing metaphase II was similar between early and late post-partum times (66.0 vs. 73.9%). Also, no differences were found in the percentage of oocytes with peripheral cortical granule migration (16.4 vs. 17.0%), rate of oocytes with partially migrated pattern (25.4 vs. 34.0%), homogeneous CG distribution (37.3 vs. 34.0%) and the percentage of abnormal oocytes with an altered pattern of CG migration (20.9 vs. 12.8%).

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4. Discussion

The semi-intensive reproductive rhythm (AI at 11 dpp) is commonly used on commercial rabbit farms (FAO, 1997), where does are generally inseminated mid- lactation. This implies depression of several reproductive features like sexual receptivity, ovulation, fertilization, implantation and embryo survival due to the negative influence of prolactin and the negative energy balance of does (for review, see

Castellini et al., 2010). Moreover, one of the non-nutritional factors having the largest impact on milk yield is the number of suckling kits (Maertens et al., 2006). The use of an extensive reproductive rhythm combined with early weaning has been proposed to improve animal welfare (Castellini, 2007). In the present work, rabbits belonging to the extensive group (25 dpp) were not weaned. Although early weaning at that time improves maternal energy reserves, it also increases kit mortality (Rebollar et al., 2009), and no significant improvements have been observed at the ovarian level compared to lactating animals as we showed in the first experiment. In the current work, a loss of body reserves throughout the post-partum period and similar ovarian features using either reproductive management are observed. Therefore, from the energetic point of view, insemination at day 25 pp would not be an adequate strategy in primiparous lactating rabbit as females seem to maintain energy reserves until day 11 pp, but due to the high intensity of lactation (8 pups/doe) and the progressive increase of milk production, body energy content is substantially lower at day 25 of lactation.

In the current study, body weight and some parameters of body composition such as body lipid depots diminished during the post-partum period. In the first experiment, we observed similar values of estimated body composition in lactating

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CHAPTER 3 primiparous does at 25 dpp. According to our findings, Feugier and Fortun-Lamothe

(2006) also found lower lipid stores in females using an extensive rhythm (AI at 25dpp) compared to a semi-intensive one (AI at 11dpp). In addition, Pascual et al. (2001) showed that the perirenal fat thickness of does was maintained during the first weeks of lactation, but it decreased considerably from 21 to 28 days, especially in non-pregnant does. It is well known, that lactation is the most energetically expensive event of the reproductive cycle in mammals (Feugier and Fortun-Lamothe, 2006). Energy losses continue through lactation (Xiccato, 1996), as has been also demonstrated in the present work.

No recovery was observed during the final phase due to the milk production for growing litters, which remains high even after 25 or 30 days of lactation (Xiccato,

1996). Parigi-Bini et al. (1996) indicated that the body reserves of does at 28 dpp were not completely recovered (energy deficit of 16%). During the final lactation period, milk dry matter and lipid concentrations increase from day 20 onwards when production begins to fall (Lebas, 1971 and 1972; Pascual et al., 1999). In the current work, water body content was higher in 25 dpp rabbit does than 11 dpp; probably, it was due to high milk content in mammary glands at that time. Concerning the weekly milk yields of does, the highest quantity is produced in the third week of lactation (Lebas, 1968;

Fernández-Carmona et al., 2006). On the other hand, body ash and protein contents increase. High foetal protein turnover has been described in the late pregnancy (Young,

1979). In addition, primiparous rabbits are still growing and the average body growth feeding a standard commercial diet has been established in 37.7 g/day (Rommers et al.,

2001).

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In our study, serum NEFA concentrations increased at parturition compared to subsequent post-partum times, as reported previously in primiparous rabbit does (Arias-

Alvarez et al., 2009). The elevation of blood NEFA concentrations as parturition approaches has also been demonstrated in cows (Chagas et al., 2007) and linked to high mobilization of triacylglycerol from adipose tissue to support the onset of greater milk yield and component demands for energy body reserves used during late pregnancy to finalize fetal growth. The NEB at parturition is even more pronounced by the reduction of feed intake at the end of pregnancy, therefore NEFA are elevated (Walter et al., 2002;

Leroy et al., 2005). Low maternal serum glucose at parturition can be due to the high requirements of glucose for final foetal growth as well (Castillo et al., 2005). Later, during the transition from pregnancy to lactation, their voluntary feed intake increases

(150 g/d in pregnancy to 400 g/d at the end of lactation peak) and consequently higher, but physiologically normal, glucose concentrations were seen (Melillo, 2007).

A decrease in blood NEFA concentrations from parturition period to 11 dpp were observed as previously reported by Arias-Álvarez et al. (2009). However, the does cannot eat enough to cover the high-energy requirements for milk production, as shown by the reduction of body lipid depots and circulating fatty acid concentrations.

However, blood NEFA levels do not reflect changes in energy balance caused by reproductive rhythm in rabbits, in agreement with Xicatto et al. (2005) because of the individual variations. Also, Arias-Álvarez et al. (2009) found lower serum protein levels at parturition, as in our study. As no change was found in serum albumin, the significant increase may reflect an increase in serum globulins or a physiological dehydration due to lactation status. Nevertheless, serum protein and albumin concentrations are within physiological ranges (Melillo, 2007).

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Lactation negatively influences all reproductive functions in does (Fortun and

Bolet, 1995; Arias-Alvarez et al., 2009 and Castellini et al., 2010) possibly due to high prolactin concentrations (McNeilly et al., 1982). Also, a direct response of the ovary

(follicles, corpus luteum) and uterus to circulating metabolites including fatty acids, amino acids, and glucose has been demonstrated in ruminants (Webb et al., 2003).

However, there is much controversy regarding the role of reproductive management in rabbit reproductive outcomes and little data is available on how management influences ovarian parameters; hence the need for the present study. Serum estradiol levels have been used as a marker of antral follicular growth and steroidogenesis (Wallach and

Noriega, 1970). In our study, steroid concentrations did not change among groups but lactating rabbits show variable serum oestradiol concentrations during the post-partum period (Ubilla and Rebollar, 1995). Reduced premating concentrations of oestradiol in rabbits inseminated on day 1 pp with respect to those inseminated on day 14 pp have been reported by Lamb et al. (1988). There was no influence of the reproductive management on follicular population categorization, nor on nuclear and cytoplasmic maturation rate which was similar to that reported in the first experiment, being nuclear maturation rate similar in the 25 dpp group (66 and 67% respectively). However, the mean preovulatory follicles counted on the ovarian surface were higher for females from the semi-intensive group than for the extensive one, as in the first experiment. In both studies, 25 dpp females had poor body condition and follicular growth was probably affected (Diskin et al., 2003). In addition, in the current work, ovaries from the

25 dpp does tended to have a slightly higher rate of atretic follicles and less healthy ones. Arias-Álvarez et al. (2009) also found more atretic follicles in ovaries of rabbits with a negative energy balance. They demonstrated that poor body reserves and lactation status in primiparous rabbits negatively affect oocyte quality and endocrine

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CHAPTER 3 parameters. Regarding reproductive performance, Theau-Clement et al. (2000) indicate that fertility and prolificacy rates of primiparous lactating females at 12 and 19 dpp are similar. However, Szendro et al., (2008) reported fewer parturitions and number of kits born per year in primiparous lactating 25 dpp inseminated females than in semi- intensive ones. Contrarily, a previous report by our group (Rebollar et al. 1994) has demonstrated higher conception and prolificacy rate in 25 dpp inseminated rabbits than

11dpp inseminated ones. Also, Feugier and Fortun-Lamothe (2006) observed higher sexual receptivity and lower foetal mortality in lactating rabbits under an extensive rhythm compared to semi-intensive management although no differences were reported in pregnancy rate and embryonic mortality. In any case, the reproductive features of rabbits were probably mainly affected by lactation condition in the two rhythms used in the current study. That could indicate that ovarian function of primiparous lactating rabbits is negatively affected whatever the post-partum period studied (semi-intensive or extensive) when the intensity of lactation is high (eight young rabbits per litter), according to their poor body reserves, that are especially low on day 25 pp.

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6. References

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Diskin, M. G., Mackey, D. R., Roche, J. F. & Sreenan, J. M., 2003. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Anim. Reprod. Sci., 78, 345-370. FAO., 1997. http://www.fao.org/docrep/t1690E/t1690E00.htm. Fernández-Carmona, J., Blas, E., Cervera, C. & Pascual, J. J., 2006. The measure of milk rabbit. World Rabbit Sc., 14 (sp), 58. Feugier, A. & Fortun-Lamothe, L., 2006. Extensive reproductive rhythm and early weaning improve body condition and fertility of rabbit does. Anim. Res., 55, 459-470. Fortun, L. & Bolet, G., 1995. Les effets de la lactation sur les performances de reproduction chez la lapine. INRA Prod. Anim., 8, 49-56. García-García, R. M., Arias-Álvarez, M., Rebollar, P. G., Revuelta, L. & Lorenzo, P. L., 2009. Influence of different reproductive rhythms on serum estradiol, testosterone levels, features of follicular population and atresia rate, and oocyte maturation in controlled suckling rabbits. Anim. Reprod. Sci., 114, 423-433. Gidenne, T. & Lebas, F., 2006. Feeding behaviour in rabbits. In: Bels V. (Ed.), Feeding in domestic vertebrates. From structure to behaviour. CABI publishing, Wallingford, UK, pp. 179-209. Hutt, K. J., McLaughlin E. A. & Holland M. K., 2006. Primordial follicle activation and follicular development in the juvenile rabbit ovary. Cell Tissue Res., 326, 809- 822. Jorritsma, R., Wensing, T., Kruip, T. A. M., Vos, P. L. A. M. & Noordhuizen J. P .T. M. L., 2003 Metabolic changes in early lactation and impaired reproductive perfomance in dairy cows. Vet. Res., 34, 11-26. Kasuya, K., 1995. The process of apoptosis in follicular epithelial cells in the rabbit ovary, with special reference to involvement by macrophages. Arch. Histol. Cytol., 58, 257-264. Lamb, I. C., Partridge, G. G., Fuller, M. F. & Racey, P. A., 1988. Fertility of the early postpartum, lactating domestic rabbit. Theriogenol., 30, 75-82. Lebas, F., 1968. Mesure quantitative de la production laitière chez la lapine. Ann. Zootech., 18, 197-208. Lebas, F., 1971. Composition chimique du lait de lapine, évolution au cours de la traite et en fonction du stade de lactation. Ann. Zootech., 20, 185-191.

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Lebas, F., 1972. Effet de la simultanéité de la lactation et de la gestation sur les performances latières chez la lapine. Ann. Zootech., 21, 129-191. Leroy, J. L. M. R., Vanholder, T., Mateusen, B., Christophe, A., Opsomer, G., Kruif, A., Genicot, G. & Van Soom, A., 2005. Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro. Reprod., 130, 485-495. Littell, R. C., Milliken, G. A., Stroup, W. W. & Wolfinger, R. D., 1996. SAS System for mixed models. Cary, NC. SAS Institute Inc. Maertens, L., Lebas, F. & Szendrö, ZS., 2006. Rabbit milk: A review of quantity, quality and non-dietary affecting factors. World Rabbit Sc., 14, 205-230. McNeilly, A. S., Glasier, A., Jonassen, J., Howie & P. W., 1982. Evidence for direct inhibition of ovarian function by prolactin. J. Reprod. Fertil., 65,559-569 Melillo, A., 2007. Rabbit Clinical Pathology. J. Exot. Pet Med., 16, 135-145. Parigi-Bini, R., Xicato, G., Cinetto, M. & Dalle Zotte, A., 1992. Energy and protein utilization and partition in rabbit does concurrently pregnant and lactating. Anim. Prod., 55,153-162. Parigi-Bini, R., Xiccato, G., Dalle Zotte, A., Castellini, C. & Stradaioli, G., 1996. Effect of remating interval and diet on the performance and energy balance of rabbit does. In Proceedings of the 6th World rabbit Congress, F. Lebas (Ed.), AFC publ., Toulouse, France, 1, 253-258. Pascual, J. J., Cervera, C., Blas, E. & Fernández-Carmona, J., 1999. Effect of high fat diets on the performance, milk yield and milk composition of multiparous rabbit does. Anim. Sc., 68, 151-162. Pascual, J. J., 2001. Early weaning of young rabbits: a review. World Rabbit Sci., 9, 165-170. Pereda, N., 2010. Evaluación de la técnica del Análisis de Impedancia Bioeléctrica para predecir la composición corporal: aplicación en conejas sometidas a diferentes sistemas de alimentación durante la recría. Tesis Doctoral. Universidad Politécnica de Madrid. Madrid. Spain, 194. Rebollar, P. G., Alvariño J. M. R. & Ubilla, E, 1994. Grouping of rabbit reproduction management by means of artificial insemination. World Rabbit Sci. 2, 87-91. Rebollar, P. G., Bonanno, A., Di Grigoli, A., Tornambè, G. & Lorenzo, P. L., 2008. Endocrine and ovarian response after a 2-Day controlled suckling and eCG treatment in lactating rabbit does. Anim. Reprod. Sc., 104, 316-328. 118

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Rebollar, P. G., Pérez-Cabal, M. A., Pereda, N., Lorenzo, P. L., Arias-Álvarez, M. & García-Rebollar, P., 2009. Effects of parity order and reproductive management on the efficiency of rabbit productive systems. Livest. Sc., 121, 227-233. Rommers. J. M., Meijerhof, R., Noordhuizen, J. P. T. M. & Kemp, B., 2001. Effect of different feeding levels during rearing and age at first insemination on body development, body composition, and puberty characteristics of rabbit does. World Rabbit Sci., 9, 101-108. Szendrö, Zs., Gerencsér, Zs., Matics, Zs., Biró-Németh, E. & Nagy, I., 2008. Comparison of two reproductive rhythms of rabbit does. In: Proceedings of the 9th World Rabbit Congress, June 10-13, Verona, Italy. Xiccato G., Trocino A., Lukefahr S.D. (Eds.),128. Theau-Clément, M., Boiti, C., Mercier, P. & Falières, J., 2000. Description of the ovarian status and fertilizing ability of primiparous rabbit at different lactation stages. In: Proceedings of 7th World Rabbit Congress, July 4-7, Valencia, Spain. Blasco E. (Ed.), A, 259-266. Tietz, N. W., 1995. Clinical Guide to Laboratory Tests. 3rd Edition, pp 518-519. Eds. W.B. Saunders Company. Philadelphia: PA. Ubilla, E. & Rebollar, P. G., 1995. Influence of the postpartum day on plasma estradiol- 17/3 levels, sexual behaviour, and conception rate, in artificially inseminated lactating rabbits. Anim. Reprod. Sci., 38, 337-344. Walters, A. H., Bailey, T. L., Pearson, R. E. & Gwazdauskas, F., 2002. Parity-Related changes in bovine follicle and oocyte populations, oocyte quality, and hormones to 90 days. J. Dairy Sc., 85, 824-832. Wallach, E. E. & Noriega, C., 1970. Effects of local steroids on follicular development and atresia in the rabbit. Fertil. Steril., 21, 253-267. Webb, R., Nicholas, B., Gong, J. G., Campbell, B. K., Gutiérrez, C. G., Garverick H. A. & Armstrong, D. G., 2003. Mechanisms regulating follicular development and selection of the dominant follicle. Reprod. Suppl., 61, 71-90. Xiccato, G., Trocino, A.t, Boiti, C. & Brecchia, G., 2005. Reproductive rhythm and litter weaning age as they affect rabbit doe performance and body energy balance. Anim. Sci., 81, 289-296. Young, V. R., 1979. Diet as a modulator of aging and longevity. Fed. Proc., 38, 1994- 2000.

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GENERAL DISCUSSION AND CONCLUSIONS

CHAPER 4

1. General discussion

In the last 15 years, the mean productivity of rabbit farms has increased and become more homogeneous through the use of artificial insemination (AI; Castellini,

1996), cycled production (Maertens et al., 1995) and very prolific genetic strains (Bolet et al., 2004; Castellini, 2007). The replacement and the mortality rates of female per year are very high and the replaced does often show poor body condition and poor health status.

The optimization of reproductive performance is one of the main facts that assure high productivity on rabbit farms. This requires that the management practices take into account the physiology and behaviour of the animals since environmental, managerial and sanitary aspects interfere with fertility and can impair it. Indeed, reproduction could be considered a “luxury” function and the reproductive system appears able to feel whether the conditions are too severe and risky for a successful reproductive cycle (Friggens, 2003). Rabbit does can sustain lactation and gestation simultaneously but this overlap depresses several aspects of reproductive activity

(sexual receptivity, ovulation, fertilization, implantation, embryo survival) due to hormonal antagonism between prolactin and gonadotropins (Kermabon et al., 1994) and energy deficit. Semi-intensive reproductive rhythm (AI at 11 days post-partum) is widely used but does not take into consideration the physiology of lactating rabbit does

(Castellini et al., 2003) and requires treatment for oestrus synchronization.

In addition, the rabbit doe of modern strains produces a lot of milk with high energetic value, which leads to a mobilization of body fat resulting in a negative energy

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CHAPER 4 balance. In the current semi-intensive reproductive rhythms, there is an extensive overlap between lactation and gestation. The resulting energetic and hormonal antagonism reduces the fertility rate and lifespan of the doe. Strategies to optimize these parameters are necessary. With increasing animal welfare concerns, more emphasis has been shifted to developing alternative methods to hormones to synchronize estrus

(biostimulation; Theau-Clément et al., 1998). An approach that combines various strategies seems to be required to meet these objectives.

Based on this premise, the main objective of this PhD thesis has been to deepen the understanding of some mechanisms involved in the reproductive physiology of the rabbit linked to negative energy balance and lactation that may affect the estimated body composition, quality of the follicles and the competence of oocytes. In the first chapter of this thesis, the objective was to assess the influence of early weaning at 25 dpp on estimated body composition, serum metabolic and ovarian parameters of primiparous lactating does when they are reared in an extensive reproductive rhythm.

Changing the weaning age also has been an effective strategy to reduce the energy deficit in rabbit does. As Xiccato et al. (2004) stated, early weaning reduced the energy deficit by limiting the duration of lactation. Consequently, the sanitary risk of young rabbits could increase (Pascual, 2001) and if weaning is performed at 25 days kit mortality increases 3.6- fold compared to weaning performed at 35 days (Rebollar et al.,

2009).

Weaning at 25 dpp increased energy reserves; non-lactating animals had more body energy content than lactating does at 25 and 32 dpp, respectively. Feed intake and serum glucose concentration were lower in weaned does, but decreased milk production

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CHAPER 4 for seven days was enough to improve slightly their energy reserves. Consequently, body lipids and protein content were also improved in non-lactating does.

The high energy output associated with milk production at 25 and 32 dpp is not entirely compensated by feed intake and rabbit does meet this energy deficit by increasing the mobilization of body reserves and loose body energy (Xiccato et al.,

1999) which impacts negatively on reproductive performance. Body fat and fat mobilization are not independent since, for example, too thin does show less fat mobilization during lactation. Some fat mobilization is normal but it has short- and long-term consequences when it differs from an optimum rate. Does that are not able to mobilize body fat have little chance of maintaining a long productive life (Quevedo et al., 2005); at the same time, a low fat level and high fat mobilization determines low fertility (Castellini et al., 2006) and increases the risk of being eliminated (Theilghard et al., 2006).

In this sense, estimated body composition and energy changes observed in this work have confirmed previous results found on primiparous lactating rabbit does

(Xiccato et al., 2005; Fortun-Lamothe and Prunier, 1999; Feugier and Fortun-Lamothe,

2006; Castellini et al., 2006). A high energy supply is required during the final lactation period, since milk dry matter and lipid concentrations increase from Day 20 onwards when production begins falling (Lebas, 1971, 1972 and Pascual et al., 1999). We found that the similar results were obtained by (Fortun-Lamothe et al. (2002 and 1999) or

Fortun-Lamothe and Prunier (1999).

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CHAPER 4

Nevertheless, ovarian features were similar in lactating and non lactating does at

32 dpp. Only the post-partum times considered (25 or 32 dpp) had a significant effect in mean preovulatory follicles, since they were lower on Day 25 pp than on Day 32 pp.

Our results show the existence of a physiological follicular growth wave between Day

25 and 32 of the post-partum period irrespective of whether rabbit does were lactating or not and of glucose concentrations availability. Similar results we have obtained in relation of oocyte maturation rates.

On Day 32 pp, oocytes matured in vitro from ovaries of lactating or non- lactating females showed higher nuclear and cytoplasmic maturation rates than oocytes of lactating does on Day 25 pp. These results confirm again the previously described progressive improvement of reproductive performance during lactation (Ubilla and

Rebollar, 1995 and Feugier and Fortun-Lamothe, 2006).

Based on these results, we conclude that the early weaning at 25 dpp seems to improve body energy stores of primiparous does, but this fact is not well reflected on their ovarian status, which is better on Day 32 pp than on Day 25 pp, regardless of weaning time and it could be performed later.

As it has been stated in previous paragraphs, the profitability of rabbit farms has increased mainly due to improvements in management and genetic selection but several problems related to animal welfare have also occurred. The replacement and the mortality rates of female per year are very high and the replaced does often show poor body condition and poor health status. Besides the negative effect of lactation, the energy deficit and metabolic stress associated with an intensive reproduction rhythm at

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CHAPER 4 first parturition are the main factors that determine a marked reduction of fertility in high producing rabbits (Fortun-Lamothe and Prunier, 1999, Castellini et al., 2003;

Xiccato et al., 2005, Feugier and Fortun-Lamothe, 2006). In this sense, it is necessary to apply reproductive rhythms in accordance to the physiology of the females, particularly those that are in their first lactation.

One possibility could be the use of very extensive rhythms proposed by Arias-

Álvarez et al. (2009). We designed the Experiment 2, to examine an alternative extensive rhythm of insemination at 25 dpp instead of 32 dpp, to short the interval parturition-insemination, avoiding early weaning to improve kit viability and we compared it to the usual management of insemination at 11 dpp. In this way, our results showed body reserves loss throughout the post-partum period and similar ovarian features in the two reproductive managements. Therefore, from the energetic point of view, insemination at Day 25 pp would not be an adequate strategy in primiparous lactating rabbit as females seems to maintain energy reserves until Day 11 pp, but due to the high intensity of lactation (8 pups/doe) and the progressive increase of milk production, body energy content are substantially lower at Day 25 of lactation.

In the current study, body weight and some parameters of body composition such as body lipids depots diminished with increased time post-partum. In the first experiment, we observed similar values of estimated body composition in lactating primiparous does at 25 dpp. No recovery is observed during the final phase due to the milk production for growing litters, which remains high even after 25 or 30 days of lactation (Xiccato, 1996).

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CHAPER 4

Physiologically normal glucose, albumin and protein concentrations were seen

(Melillo, 2007) and blood NEFA levels did not reflect changes in energy balance caused by reproductive rhythm in rabbits in agreement with Xicatto et al. (2005) because the individual variations.

In this study, steroid concentrations did not change among groups. There was no influence of the reproductive management on follicular population categorization, neither on nuclear and cytoplasmic maturation rate which was similar to that reported in the first experiment. Nuclear maturation rate in 25 dpp group was also similar to those of the first experiment (66 and 67% respectively). However, the mean of preovulatory follicles counted on the ovarian surface were higher for females of semi-intensive group than for extensive one. In both studies, 25 dpp females presented poor body condition and probably follicular growth was affected (Diskin et al., 2003). In addition, in the current work, ovaries of 25 dpp group tended to show slight higher rate of atretic follicles and lower of healthy ones. Arias-Álvarez et al. (2009), also show more atretic follicles in ovaries of rabbits with negative energy balance. They demonstrated that poor body reserves and lactation status in primiparous rabbits negatively affect oocyte quality and endocrine parameters.

In any case, reproductive features of rabbits probably have been mainly affected by lactation condition in the two rhythms used in the current work. It could be indicating that ovarian function of primiparous lactating rabbit is negatively affected whatever it be the post-partum period studied (semi-intensive or extensive) when intensity of lactation is high (eight young rabbits per litter), according to their poor body reserves that are specially low at Day 25 pp.

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CHAPER 4

Summarizing our results of present and previous works, we conclude that it is very difficult to get good reproductive parameters in primiparous lactating rabbits. In these animals, lactation causes a high energy deficit and they are not able to feed enough to cover energy expenses; also they have not reached their adult weight and have special requirements to gain it. The implementation of more extensive reproductive rhythms should improve the productive results. However, in present work we found poor body reserves and no enhancement in ovarian features in lactating does at Day 25 pp, probably because the high intensity of lactation impaired body condition in these females. One solution to this problem could be to carry out early weaning, but we also have demonstrated that it only enhances the body reserves and no significant improvements have been found at ovary level; because it reduces feed intake and glucose availability of mothers. Consequently, extensive management for insemination of primiparous rabbit does at Day 25 pp presumably has no advantages over the semi- intensive regime of insemination at 11 dpp. From the practical point of view, although fertility is low at 11 dpp, this reproductive regime reduces interval between parturitions and avoids excessive fattening of animals not pregnant at Day 11 pp. From a physiologically and welfare vision a more extensive rhythms of insemination with a later weaning, as that proposed in Arias-Álvarez et al. (2009) study (insemination at 32 dpp and weaning at 28 dpp), would be desirable, at least, in primiparous does. Probably, the farmer must choose the best combination for their rabbit exploitation to keep this balance.

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CHAPER 4

2. General Conclusions

The general conclusions of this Thesis have been as follows:

 Early weaning at 25 dpp seems to improve body energy stores of primiparous

does, but this fact is not well reflected on their ovarian status, which is better on

Day 32 pp than on Day 25 pp, regardless of weaning time and it could be

performed later.

 Extensive management for insemination of primiparous rabbit does at Day 25 pp

presumably have no advantages over the semi-intensive regime of insemination

at 11 dpp.

 From a physiological and animal welfare vision, an extensive rhythm longer

than 25 dpp with a later weaning would be desirable, at least, in primiparous

lactating does.

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