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Theses and Dissertations Theses and Dissertations
1-1-2011
Modifying the Double-Ovsynch Protocol to include Human Chorionic Gonadotropin to Synchronize Estrus in Dairy Cattle
Joseph Andrew Binversie
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Recommended Citation Binversie, Joseph Andrew, "Modifying the Double-Ovsynch Protocol to include Human Chorionic Gonadotropin to Synchronize Estrus in Dairy Cattle" (2011). Theses and Dissertations. 3173. https://scholarsjunction.msstate.edu/td/3173
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MODIFYING THE DOUBLE-OVSYNCH PROTOCOL TO INCLUDE HUMAN
CHORIONIC GONADOTROPIN TO SYNCHRONIZE ESTRUS IN DAIRY
CATTLE
By
Joseph Andrew Binversie
A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Animal Physiology in the Department of Animal and Dairy Sciences
Mississippi State, Mississippi
August 2011 Template Created By: Damen Peterson 2009
Copyright by
Joseph Andrew Binversie
2011 Template Created By: Damen Peterson 2009
MODIFYING THE DOUBLE-OVSYNCH PROTOCOL TO INCLUDE HUMAN
CHORIONIC GONADOTROPIN TO SYNCHRONIZE ESTRUS IN DAIRY
CATTLE
By
Joseph Andrew Binversie
Approved:
______Jamie E. Larson Peter L. Ryan Assistant Professor of Associate Provost for Academic Affairs Animal and Dairy Sciences Professor of Animal and Dairy Sciences (Director of Thesis) Graduate Coordinator of Animal Physiology (Committee Member)
______Justin D. Rhinehart George Hopper Assistant Professor of Animal Science Interim Dean The University of Tennessee College of Agriculture and Life Sciences (Committee Member)
Template Created By: Damen Peterson 2009
Name: Joseph Andrew Binversie
Date of Degree: August 6, 2011
Institution: Mississippi State University
Major Field: Animal Physiology
Major Professor: Jamie E. Larson
Title of Study: MODIFYING THE DOUBLE-OVSYNCH PROTOCOL TO INCLUDE HUMAN CHORIONIC GONADOTROPIN TO SYNCHRONIZE ESTRUS IN DAIRY CATTLE
Pages in Study: 82
Candidate for Degree of Master of Science
The objectives of this study were to determine whether conception, ovulation rates, presynchronization rates, or follicle and CL characteristics were altered after modifying the Double-Ovsynch (DO) protocol to include human chorionic gonadotropin
(hCG) compared to the conventional DO protocol in dairy heifers and lactating dairy cows. We hypothesized that replacing the first injection of gonadotropin releasing hormone (GnRH) in the DO protocol with hCG would increase the proportion of females that ovulate, thus improving the presynchronization rate leading to increased conception rates. Ovulation rates were increased in cows administered hCG compared to GnRH, but subsequent synchronization, and conception rates did not differ between treatments. A greater proportion of hCG-treated cows tended to fail to have undergone luteolysis compared to those treated with GnRH. In conclusion, no improvement in overall fertility was achieved by replacing the first injection of GnRH in the DO protocol with hCG.
Key Words: Double-Ovsynch, hCG, presynchronization Template Created By: Damen Peterson 2009
DEDICATION
I would like to dedicate this thesis to my wife Liz. Thank you for all of your love and support throughout my masters program at Mississippi State University. You always are able to brighten my day.
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ACKNOWLEDGEMENTS
I would first like to thank all of the graduate students that assisted me in my research project at Mississippi State University. Without your help, this research would not have been possible. A special thank you to Kenneth Graves and JB Gardner, I know having the cows and heifers up and ready every morning was a challenge at times, and I am grateful that you guys were always able to accommodate my research needs. Thank you also to David Skinner for his assistance in the breeding of the heifers and cows.
Thank you to the Mississippi Agricultural and Forestry Experiment Station for the use of their facilities and animals and also to all the members in the Department of Animal and
Dairy Sciences for your help and support throughout my time in Mississippi. I would also like to thank my major professor Dr. Jamie Larson for allowing me the freedom to pursue a research project in the area of estrous synchronization. I believe that you have helped me become a more independent researcher and a better statistician and writer.
Thank you also to my committee members of Dr. Peter Ryan and Dr. Justin Rhinehart. I always appreciated the friendship and advisement that you both gave me. Finally, I would like to thank my family. I am very appreciative that the values of hard work and respect towards one another have helped me be the person I am today. Thank you for all of your love and support.
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TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
LIST OF TABLES ...... vi
LIST OF FIGURES ...... vii
CHAPTER
I. INTRODUCTION ...... 1
II. LITERATURE REVIEW ...... 4
Steroid Metabolism in Dairy Cattle ...... 4 Synchronization of the Estrous Cycle ...... 6 Presynchronization of the Estrous Cycle ...... 10 Presynchronization with PGF2α ...... 10 Presynchronization with CIDR devices ...... 11 Presynchronization with GnRH and PGF2α ...... 13 Characteristics of Human Chorionic Gonadotropin ...... 15 The use of hCG in Synchronization of Estrus ...... 16 The use of hCG Post-AI ...... 20 The use of hCG in Resynchronization Protocols ...... 23 The use of hCG in Heifers ...... 25 Summary ...... 27 References ...... 28
III. MODIFYING THE DOUBLE-OVSYNCH PROTOCOL TO INCLUDE HUMAN CHORIONIC GONADOTROPIN TO SYNCHRONIZE ESTRUS IN DAIRY CATTLE ...... 36
Abstract ...... 36 Introduction ...... 37 Materials and Methods ...... 39 Herd Management Practices ...... 39 Experimental Design and Treatments ...... 40 iv Template Created By: Damen Peterson 2009
Milk Production and BCS ...... 42 Ovarian Measurements and Pregnancy Diagnosis ...... 42 Blood Collection and RIA ...... 43 Statistical Analyses ...... 44 Results ...... 45 Experiment 1: Dairy Cows...... 45 Ovarian Responses to hCG or GnRH during Pre-Ovsynch ...... 45 Measurements at time of PGF2α of Pre-Ovsynch ...... 46 Responses to PGF2α and Day 10 Measurements ...... 47 Evaluation of Breeding-Ovsynch ...... 49 Experiment 2: Dairy Heifers ...... 50 Ovarian Responses to hCG or GnRH during Pre-Ovsynch ...... 50 Responses to PGF2α and Day 10 Measurements ...... 51 Evaluation of Breeding-Ovsynch ...... 52 Discussion ...... 53 Conclusions ...... 64 References ...... 66
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LIST OF TABLES
1. Plasma concentrations of progesterone and ovarian measurements taken in all cows during Pre-Ovsynch (d 0, 7, and 10) ...... 73
2. Subset of cows that ovulated to injection of hCG or GnRH (d 0) and their plasma progesterone and ovarian measurements during Pre-Ovsynch (d 0, 7, and 10) ...... 74
3. Ovulation, luteal regression, and synchronization rates in all cows and subset of cows that ovulated to hCG or GnRH injection during Pre-Ovsynch ...... 75
4. Conception rates and characteristics of progesterone of all cows measured during Breeding-Ovsynch...... 75
5. Effect of parity on largest follicle diameter located on either ovary on d 0, 7, and 10, and concentrations of progesterone on d 17 ...... 76
6. Plasma concentrations of progesterone and ovarian measurements taken in heifers during Pre-Ovsynch (d 0, 7, and 10) ...... 77
7. Luteal regression, synchronization, and conception rates in heifers ...... 78
8. Effect of age and breed of heifer on concentrations of progesterone, ovarian measurements, and synchronization and conception rates ...... 79
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LIST OF FIGURES
1. Schematic of treatment protocols and sampling schedule for cows and heifers during Double-Ovsynch ...... 80
2. Concentrations of progesterone (P4) on d 10 of Pre-Ovsynch in dairy cows (LSMeans ± SEM; Experiment 1) ...... 80
3. Conception rates at d 32 post-TAI by treatment and AI technician in dairy cows (Experiment 1) ...... 81
4. Diameter of largest follicle located on either ovary on d 7 of Pre- Ovsynch in heifers (LSMeans ± SEM; Experiment 2) ...... 82
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CHAPTER I
INTRODUCTION
Previous to the mid-1990s, dairy cattle producers had to perform estrus detection several times a day to determine the appropriate time to inseminate their cows. In 1994, it was estimated that the failure and inaccuracy of heat detection resulted in an annual loss of $300 million to the dairy industry in the United States (Senger, 1994). This was due to the fact that most dairy herds have an estrus detection efficiency of less than 50%
(Senger, 1994; Washburn et al., 2002). Investigation into the cause of decreased estrus detection rates and fertility in dairy cows suggest that, in part, increased feed intake from high producing dairy cows results in increased metabolic clearance and thus decrease of circulating reproductive steroid hormones necessary for normal reproductive functions
(Sangsritavong et al., 2002; Wiltbank et al., 2006; Rhinehart et al., 2009).
However, over the past 10 to 15 years estrus synchronization protocols have been developed to combat these ever growing problems. These protocols, referred to as timed
AI (TAI) protocols, allow dairy producers to use pharmaceutical technologies to manipulate a cow’s reproductive cycle so that insemination can take place on a specific day of their choice. Their use greatly reduces the need for estrus detection and increases the overall reproductive efficiency of the herd (Pursley et al., 1997a). Greater conception
1 Template Created By: Damen Peterson 2009 and pregnancy rates make a protocol more profitable for dairy producers (De Fries,
2007).
The estrus synchronization protocol referred to as Ovsynch was introduced to the dairy industry in the mid to late 1990s (Pursley et al., 1995; Pursley et al., 1997b). Since then, researchers have developed several modifications to the original Ovsynch (Geary and Whittier, 1998; Pursley et al., 1998; Moreira et al., 2001; El-Zarkouny et al., 2004;
Brusveen et al., 2008). Conception rates are greatest when the Ovsynch protocol is initiated on d 5 to 12 of the estrous cycle (Vasconcelos et al., 1999). This results in greater rates of ovulation to the first injection of gonadotropin releasing hormone (GnRH) of Ovsynch and thus increased conception rates (Vasconcelos et al., 1999; Cartmill et al.,
2001). Therefore, a critical component for successful synchronization of ovulation in dairy cattle involves the inclusion of a presynchronization stage (Moreira et al., 2001;
Navanukraw et al., 2004; Bello et al., 2006; Galvão et al., 2007). This presynchronization stage increases the likelihood of ovulation to the first injection of
GnRH of Ovsynch, leading to increased responsiveness to subsequent hormone injections and thus increased conception rates (Bello et al., 2006).
Souza et al. (2008) introduced the novel idea of putting two Ovsynch protocols together to form what is known as Double-Ovsynch. The first Ovsynch is referred to as
Pre-Ovsynch and is used for presynchronizing the follicular growth. Seven days after the
Pre-Ovsynch, another Ovsynch, the Breeding-Ovsynch, is initiated and the cow is inseminated after this Ovsynch. In this study, human chorionic gonadotropin (hCG) was utilized in an attempt to improve the rate of synchrony to the Pre-Ovsynch of Double-
Ovsynch. Human chorionic gonadotropin is a hormone that has similar activity to
2 Template Created By: Damen Peterson 2009 luteinizing hormone (LH), inducing ovulation by binding to LH receptors on the follicle and producing LH-like effects (Stevenson et al., 2007). Therefore, with the use of hCG, ovulation of a dominant follicle is no longer dependent upon the LH surge produced from an injection of GnRH. Research has reported that hCG has an increased capacity to induce ovulation when compared to GnRH (Stevenson et al., 2007; Dahlen et al., 2008;
Buttrey et al., 2010; Dahlen et al., 2011). Further investigation is warranted to determine the efficacy of hCG in the presynchronization stage of Double-Ovsynch.
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CHAPTER II
LITERATURE REVIEW
Steroid Metabolism in Dairy Cattle
For many years, dairy cattle in the United States have been genetically selected for increased milk production (Cunningham 1983; Boettcher et al., 1993). A dramatic decrease in reproductive performance and efficiency has also occurred during this same time period (Butler and Smith, 1989; Butler, 1998; Lucy, 2001). Butler (1998) reported an inverse relationship between the decline in first-service conception rates (65% to 40%) and annual milk production (4500 to 9000 kg/yr) from 1951 to 1996 for Holstein dairy cows in New York. Likewise, Washburn et al. (2002) reported that Holstein dairy cattle in the Southeastern United States from 1976 to 1999 increased in average days open (124 to 168 d) and in services per conception (1.91 to 2.94). A general decline in estrus detection rates (50.9 to 41.5%) was also noted (Washburn et al., 2002). Investigation into the cause of this decline has led to the hypothesis that there is a negative relationship between milk production and reproductive performance in dairy cattle (Hansen, 2000;
Lucy, 2001; Lopez et al., 2004). Several theories have been proposed to explain this and are still being examined. Research suggests that one of the major factors attributing to this decline is increased feed intake of high producing cows which, in turn, results in negative reproductive physiological conditions (Wiltbank et al., 2006).
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When considering the current physiological conditions facing the modern dairy cow as related to reproductive efficiency, steroid metabolism is an important concept. It has been proposed that some of the negative reproductive changes observed in lactating dairy cows are caused by dramatic increases in the metabolism of steroid hormones due to increased consumption of feed and blood flow to the liver (Sangsritavong et al., 2002;
Wiltbank et al., 2006). Prior to feeding, hepatic blood flow was greater in lactating cows
(1561 ± 57 L/h) than in similar size and age, non-lactating (747 ± 47 L/h) cows
(Sangsritavong et al., 2002). Also in that study, hepatic blood flow and metabolism of progesterone and estrogen increased immediately after any amount of feed was consumed in both lactating and non-lactating cows. However, lactating cows had over twice the metabolism of estrogen and progesterone compared to non-lactating cows. It is important to note that dry matter intake and milk production are highly correlated, with an R-value of 0.88 (Harrison et al., 1990). So, as milk production increases, one can expect feed consumption to increase as well.
Lopez et al. (2004) determined the correlation between milk production and duration of estrus and other reproductive parameters in lactating dairy cows. There was a significant decrease in duration of estrus in high (46.8 kg/day) compared to low (32.3 kg/day) producing cows (6.2 and 10.9 h, respectively). Additionally, there were significantly decreased concentrations of estradiol during estrus when comparing high and low producers (6.8 and 8.6 pg/mL, respectively). Interestingly, high producing cows had significantly larger dominant follicles when compared to the lower producing cows
(18.6 and 17.4 mm, respectively). Based upon these data, the decreased concentrations of estradiol could also have caused increased follicular size by delaying the time at which
5 Template Created By: Damen Peterson 2009 the onset of estrus occurs. Because of this delay, the GnRH and LH surge would occur later than normally and result in ovulation of an aged oocyte from a larger dominant follicle and thus compromise its fertility (Wiltbank et al., 2006).
Since dry matter intake increases in high producing cows, an increase in blood flow to the digestive tract and liver will result in an increase in the metabolism of steroid hormones such as estradiol and progesterone (Sangsritavong et al., 2002). Once this occurs, there will be decreased circulating concentrations of estradiol and progesterone.
With this decrease in reproductive steroid hormones, negative reproductive consequences are then likely to occur. These include decreased conception rates, increased pregnancy loss, increased multiple ovulation rates (resulting in an increased twinning rate), and decreased behavioral estrus (Wiltbank et al., 2006). Considering this knowledge, the need for estrus synchronization to combat these reproductive deficiencies becomes increasingly more evident. Proper estrus synchronization can eliminate the need for estrus detection, and increase deficient endogenous steroid hormones such as progesterone (El-Zarkouny et al., 2004; Bello et al., 2006; Cunha et al., 2008; Souza et al., 2008) and estradiol (Pancarci et al., 2002; Cerri et al., 2004; Souza et al., 2007).
Synchronization of the Estrous Cycle
The estrus synchronization protocol known as Ovsynch (Pursley et al., 1995) was developed during the mid to late 1990s to help dairy producers better manage the reproductive programs for their herds as well as combat decreasing estrus detection rates.
Ovsynch begins with an injection of GnRH given to cause ovulation of a dominant follicle and result in the formation of a corpus luteum (CL). A new follicular wave will
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begin and 7 d later, prostaglandin F2α (PGF2α) is given to regress the new CL. This is followed either 48 (Pursley et al., 1995) or 56 h (Brusveen et al., 2008) later with a second injection of GnRH and finally TAI 16 h after the final injection of GnRH (Pursley et al., 1998).
Although TAI was a major breakthrough for the dairy industry, the original
Ovsynch protocol did have deficiencies. The major drawback to consider when the first injection of GnRH is given to induce ovulation of a dominant follicle is that the stage of its estrous cycle is completely random. Because of this, the Ovsynch protocol is most effective when the first injection of GnRH is given on d 5 to 12 of the estrous cycle
(Vasconcelos et al., 1999; Moreira et al., 2000a; Cartmill et al., 2001; Moreira et al.,
2001). Initiation of Ovsynch in metestrus (d 1 to 4 of the estrous cycle) often results in failure of a new follicular wave due to lack of ovulation (Vasconcelos et al., 1999;
Moreira et al., 2000a), while initiation in late diestrus (d 13 to 17 of the estrous cycle) results in premature estrus before the second injection of GnRH caused by premature CL regression (Moreira et al., 2000a). Moreover, initiation of the Ovsynch protocol during proestrus (d 18 to 21 of the estrous cycle) results in incomplete luteal regression from the injection of PGF2α (Moreira et al., 2000a). Incomplete luteal regression compromises fertility by significantly reducing conception rates as compared to complete luteolysis
(Moreira et al., 2000b; Souza et al., 2007; Brusveen et al., 2009).
Specifically, the greatest ovulation rate to the first injection of GnRH in the
Ovsynch protocol was achieved on d 5 to 9 (96%) as compared to d 1 to 4 (23%), d 10 to
16 (54%), and d 17 to 21 (77%) of the estrous cycle (Vasconcelos et al., 1999). Cows ovulating a follicle to the first injection of GnRH of Ovsynch were significantly more
7 Template Created By: Damen Peterson 2009 likely to ovulate to the second injection of GnRH of Ovsynch (92 and 79%), which is required for successful conception. When comparing cows in the first half of the estrous cycle (d 1 to 12) to the second half of the estrous cycle (d 13 to 22) at the initiation of
Ovsynch, a significantly greater proportion of cows in the first half of the estrous cycle were synchronized to Ovsynch (luteal regression from PGF2α and ovulation to final
GnRH) as compared to cows in the second half of the estrous cycle (91 and 80%, respectively; Vasconcelos et al., 1999). Collectively, initiation of the Ovsynch protocol on d 5 to 12 of the estrous cycle achieved the greatest conception rates and should be the target for successful synchronization of ovulation.
One slight modification to the original Ovsynch protocol was to administer AI and an injection of GnRH concurrently either 48 (CO-Synch-48; Geary and Whittier,
1998), or 72 h (CO-Synch-72; Portaluppi and Stevenson, 2005) after the injection of
PGF2α. Rationale for the CO-Synch protocol was to achieve comparable conception rates to Ovsynch but decrease labor costs because cattle (particularly beef cattle) would be handled only three times as compared to four times in the Ovsynch protocol (Geary and
Whittier, 1998; Portaluppi and Stevenson, 2005). Comparisons of protocols indicate that
Ovsynch generally has increased conception rates over CO-Synch (Pursley et al., 1998;
Geary and Whittier, 1998; Brusveen et al., 2008) but not so in one particular study
(Portaluppi and Stevenson, 2005).
Supplementation of progesterone using a controlled internal drug release (CIDR) device has also been incorporated into the basic Ovsynch protocol (El-Zarkouny et al.,
2004; Stevenson et al., 2006), with it typically being administered at the time of the first injection of GnRH and then removed 7 d later at the time of the injection of PGF2α.
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Circulating concentrations of progesterone prior to AI have been reported to be positively correlated with increased conception rates (Fonseca et al., 1983). Therefore, supplementation of progesterone from the CIDR could potentially increase conception rates of anovular cows deficient in progesterone (El-Zarkouny et al., 2004; Stevenson et al., 2006). Cows receiving the Ovsynch + CIDR protocol had significantly increased conception rates compared to Ovsynch (59.3 and 36.3%) at d 29 post-TAI (El-Zarkouny et al., 2004). In addition, anovular cows at the onset of the TAI protocols had increased conception rates when treated with Ovsynch + CIDR as compared to Ovsynch (64 and
27%). In comparison, no difference in conception rate was detected between cycling cows for the Ovsynch or Ovsynch + CIDR protocols (47 and 54%).
Interestingly, inclusion of a CIDR in the Ovsynch protocol has been reported to be beneficial in some studies (El-Zarkouny et al., 2004; Melendez et al., 2006; Stevenson et al., 2006; Chebel et al., 2010) but not all (Galvão et al., 2004; Stevenson et al., 2008a).
Chebel et al. (2010) reported no added benefit of using a CIDR in anovular cows, but did see an overall significant increase in conception rates when comparing all cows (cyclic and noncyclic) that were treated with the Ovsynch + CIDR protocol compared to
Ovsynch (38.1 and 33.3%, respectively). It was concluded that the added benefit for the inclusion of a CIDR in the Ovsynch protocol in their study mostly benefited cyclic cows that would have otherwise undergone early luteal regression before the injection of PGF2α in the Ovsynch protocol, thus compromising the efficacy of the protocol and subsequent conception rates (Chebel et al., 2010).
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Presynchronization of the Estrous Cycle
Presynchronization with PGF2α
After determining the proper stage of the estrous cycle to initiate the Ovsynch protocol (Vasconcelos et al., 1999), presynchronizing cows to increase ovulation rates to the first injection of GnRH of Ovsynch became a major focal point of research. Moreira et al. (2001) administered two injections of PGF2α, 14 d apart followed 12 d later by the first injection of GnRH for the Ovsynch protocol. Results indicated that the two injections of PGF2α significantly increased the conception rates for cyclic cows when compared to the normal Ovsynch protocol (46.9 and 34.4%). This protocol became known as Presynch-12, with the 12 corresponding to the 12 d between the second injection of PGF2α and the onset of Ovsynch. Two other presynchronization protocols
(Presynch-14 and Presynch-11) involving two injections of PGF2α given 14 d apart were also developed (Navanukraw et al., 2005; Galvão et al., 2007). All three presynchronization protocols significantly increase conception rates compared to the basic Ovsynch protocol (Moreira et al., 2001; Navanukraw et al., 2004; Galvão et al.,
2007). The Presynch-11 protocol caused a significant increase in ovulation rates to the first injection of GnRH when compared to Presynch-14 (61.4 and 44.7%) as well as a significant increase in conception rates ( 40.5 and 33.5%; Galvão et al., 2007). Thus as ovulation rates increase to the first injection of GnRH of Ovsynch, conception rates increase as well.
Recently, five protocols for presynchronization including Presynch-14, Presynch-
12, and Presynch-10 before the initiation of a CO-Synch TAI protocol (GnRH-7 d-
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P G F 2 α -3 d- Gn R H+ T AI) were co mpared (Stevenson, 2011). It was hypothesized that d e cr e asi n g t h e i nt er v al fr o m t h e s e c o n d i nj e cti o n of P G F 2 α of Pr es y n c h t o t h e first injection of Gn R H of C O-Synch fro m 14 to 12 to 10 d, a greater proportion of co ws w o ul d o v ul at e t o t h e first i nj e cti o n of G n R H of C O- S y n c h. It w as pr e di ct e d t h at c o ws i n the Presynch-14 group would be at d 9 to 14 of the estrous cycle, while Presynch-12 c o ws w o ul d b e at d 7 t o 1 2 a n d Pr es y n c h- 1 0 c o ws at d 5 t o 1 0. S ur prisi n gl y, t h er e w as n o si g nifi c a nt diff er e n c e i n o v ul ati o n r at es b et w e e n tr e at m e nt gr o u ps at t h e i niti ati o n of t h e
C O - S y n c h pr ot o c ol or i n c o n c e pti o n r at es. H o w e v er, o v ul ati o n r at es at t h e o ns et of C O-
Synch were significantly greater in co ws with elevated progesterone (≥ 1 ng/ m L) and larger follicle size ( ≥14 m m). Although no differences were reported bet ween the three
Pr es y n c h pr ot o c ols, o v ul ati o n t o t h e first i nj e cti o n of G n R H of C O- S y n c h si g nifi c a ntl y i n cr e as e d c o n c e pti o n r at es ( St e v e ns o n, 2 0 1 1).
Presynchronization with CI D R devices
The greatest obstacle encountered with standard presynchronization protocols is t h at P G F 2 α d o es n ot aff e ct a n o v ul ar c o ws. T h at is, c o ws t h at ar e n ot c ycli n g a n d d o n ot h a v e a C L pr es e nt o n a n o v ar y will n ot r es p o n d t o t h e i nj e cti o ns of P G F 2 α a n d t h us n ot o v ul at e. T y pi c all y, 2 0 t o 3 0 % of d air y c o ws 6 5 t o 7 5 d i n mil k ar e a n o v ul ar ( Gü me n et al., 2 0 0 3; L o p e z et al., 2 0 0 5). I ns erti o n of a CI D R i n t h e pr es y n c hr o ni z ati o n st a g e m a y pr o m ot e c ycli cit y i n a n o v ul ar c o ws ( C h e b el et al., 2 0 0 6; Bi c al h o et al., 2 0 0 7; Br u n o et al., 2 0 0 9; C erri et al., 2 0 0 9). C h e b el et al. ( 2 0 0 6) i n cl u d e d t h e us e of a CI D R i nt o a
Presynch-13 protocol (P GF 2 α -7 d- CI D R inserted-7 d- P GF 2 α + CI D R re moval-13 d-
Ovsynch) and reported that a significantly greater proportion of co ws previously
1 1 Template Created By: Damen Peterson 2009 established as anovular resumed cyclicity by the onset of Ovsynch when compared to
Presynch-13 cows not receiving a CIDR (48 and 31%, respectively). However, conception rate was not improved with the inclusion of a CIDR. Modifying a Presynch-
14 protocol to include a CIDR insert for 7 d similar to Chebel et al. (2006) also decreased the proportion of cows with low progesterone (< 1 ng/mL) at the onset of Ovsynch
(Bicalho et al., 2007). However, conception rates were not improved (Bicalho et al.,
2007).
Further studies were conducted to establish efficacy of a CIDR administered for 7 d during the Presynch-12 (Cerri et al., 2009) and Presynch-11 (Bruno et al., 2009) protocols, but conception rates were not improved through the addition of a CIDR insert.
However, improvement in ovulation rates to the first injection of GnRH of Ovsynch for
CIDR-treated cows was reported (Bruno et al., 2009). Stevenson (2011) compared two
CIDR presynchronization protocols referred to as CIDR-10 (CIDR inserted-7 d-CIDR removed + PGF2α-10 d-CO-Synch-72) and CIDR-3 (CIDR inserted-7 d-CIDR removed +
PGF2α-3 d-CO-Synch-72) to a control CO-Synch protocol. It was hypothesized that the
CIDR-10 protocol would have a greater conception rate over CIDR-3 due to previous literature reporting the significance of increased (≥ 1 ng/mL) circulating concentrations of progesterone at the onset of Ovsynch (Cunha et al., 2008; Galvão and Santos, 2010).
As expected, concentrations of progesterone were significantly greater in the CIDR-10 and CO-Synch protocols at the first injection of GnRH of CO-Synch as compared to
CIDR-3 (3.0, 3.7, and 0.9 ng/mL). Likewise, conception rates for the CIDR-10 protocol
(36%) was significantly greater than CIDR-3 (16%) but was similar to the control CO-
Synch protocol (34%). With limited success through the incorporation of a CIDR in the
12 Template Created By: Damen Peterson 2009 presynchronization stage, and no clear evidence of an increase in conception rates, focus shifted towards the inclusion of GnRH and PGF2α in presynchronization protocols.
Presynchronization with GnRH and PGF2α
The first successful attempt to incorporate GnRH and PGF2α into a presynchronization protocol was Bello et al. (2006). In this study there were three different treatment groups with an Ovsynch group serving as the control. The treatment protocol referred to as G6G (PGF2α-2 d-GnRH-6 d-Ovsynch) was reported to be the most successful of the treatment groups. The G6G protocol caused a significant increase in ovulation rates to the first injection of GnRH in the Ovsynch portion of the protocol when compared to the standard Ovsynch (85 and 54%, respectively). The overall synchronization rate of all the groups was then determined by luteal regression from the last injection of PGF2α and ovulation to the final injection of GnRH. The G6G protocol caused a significant increase in synchronization rate when compared to the basic
Ovsynch protocol (92 and 69%, respectively). Conception rates tended to be increased in the G6G group when compared to Ovsynch (50 and 27%). Although the G6G protocol showed promising results, this protocol is rarely used in the dairy industry because dairy producers would have to handle their cows 5 d a wk. For many dairy producers, that requirement of time and labor is unacceptable.
A recently developed estrus synchronization protocol known as Double-Ovsynch has significantly increased conception rates over the standard Presynch protocol and proven to be more practical than G6G (cows handled 4 d a wk compared to 5). Souza et al. (2008) introduced the novel idea of putting two Ovsynch protocols together to form
13 Template Created By: Damen Peterson 2009 what is known as Double-Ovsynch. The first Ovsynch is referred to as Pre-Ovsynch and is used for presynchronizing follicular growth. After the Pre-Ovsynch, another Ovsynch, the Breeding-Ovsynch, is initiated 7 d later and the cow is inseminated at the conclusion.
When the Presynch-12 protocol was compared to Double-Ovsynch, there was a significantly greater proportion of cows in the Presynch-12 group with low progesterone
(< 1 ng/mL) at the onset of Ovsynch as compared to the Double-Ovsynch group (33.3 and 9.4%; Souza et al., 2008). This difference can most likely be attributed to the reduction in anovular cows through the use of GnRH in the Double-Ovsynch presynchronization stage. Overall conception rates were also significantly increased for the Double-Ovsynch protocol when compared to Presynch-12 (49.7 and 41.7%).
In addition, circulating concentrations of progesterone were significantly increased at the time of the last injection of PGF2α before the cow was to be inseminated.
Double-Ovsynch had a significantly greater proportion of cows with increased concentrations of progesterone (≥ 3 ng/mL) at this point over Presynch-12 (78.1 and
52.2%, respectively). Increased concentrations of progesterone during follicular growth in the Double-Ovsynch protocol as compared to Presynch-12 was likely due to a greater proportion of cows in the Double-Ovsynch treatment having two CLs at this point.
Further investigation is needed to determine whether or not the increase in conception rates from Double-Ovsynch are a result of increased efficacy of the presynchronization stage or perhaps in combination with an increase in progesterone during follicular development.
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Characteristics of Human Chorionic Gonadotropin
Human chorionic gonadotropin (hCG) is a hormone that has been used sporadically by dairy producers for the past several years. Recently, interest in this hormone for use in the synchronization of estrus seems to have increased not only from dairy producers but researchers as well. Human chorionic gonadotropin is a large glycoprotein molecule (~ 37 kDa) and has a half-life of approximately 30 h and thus will stay in circulation for a prolonged period of time (De Rensis et al., 2010). Heifers treated with 3000 IU of hCG (1000 IU i.v. and 2000 IU i.m.) had significantly increased circulating concentrations of hCG 30 h post-treatment and had not returned to basal concentrations by 66 h post-treatment (Schmitt et al., 1996). In comparison, the LH- induced surge from the injection of GnRH lasts only for about 5 h until returning to basal concentrations (Chenault et al., 1990). Human chorionic gonadotropin also has similar activity to luteinizing hormone (LH), inducing ovulation by binding and stimulating LH receptors on the follicle (Stevenson et al., 2007). Therefore, ovulating a dominant follicle with hCG circumvents the need for a GnRH-induced LH surge produced from an injection of GnRH.
Stevenson et al. (2007) reported that hCG induced a greater ovulation rate compared to GnRH when given from d 4 to d 9 post-AI (78 and 60%). In this study, both
GnRH and hCG induced accessory CLs after injection, with a 25.5% incidence of multiple ovulations and hCG accounting for 76.9% of multiple ovulations as compared to
23.1% for GnRH. When compared, GnRH typically ovulated a single dominant follicle when administered, whereas hCG had more often ovulated the dominant follicle as well as a subordinate follicle (Stevenson et al., 2007). Moreover, administration of hCG
15 Template Created By: Damen Peterson 2009 causes a greater increase in progesterone when compared to GnRH (Stevenson et al.,
2007). This is thought to be a result of not only the induction of accessory CL but also by hCG increasing endogenous concentrations of progesterone from an existing CL
(Stevenson et al., 2007). It has been theorized that hCG could potentially have greater efficacy over GnRH in high producing, subfertile cows where the concentration of LH during the surge may be compromised and result in incomplete ovulation (De Rensis et al., 2010).
The use of hCG in Synchronization of Estrus
Use of hCG in the synchronization of estrus thus far has produced variable results,
(De Rensis et al., 2002; Burns et al., 2008; De Rensis et al., 2008a,b; Marquezini et al.,
2009). In Burns et al. (2008), hCG replaced the first injection of GnRH in the CO-Synch
+ CIDR protocol. Beef cattle received either an injection of GnRH or hCG along with a
CIDR for 7 d. After 7 d, PGF2α was administered at the time of CIDR removal followed
62 h later by an injection of GnRH concurrent with TAI. Cows treated with hCG had significantly reduced pregnancy rates compared to cows treated with GnRH (38.9 and
53.4%), with the greatest reduction in cycling cows. The mechanism of action causing this reduction is currently unknown (Burns et al., 2008).
Keskin et al. (2010) incorporated hCG into the basic Ovsynch protocol by also replacing the first injection of GnRH with hCG in dairy cows. It was hypothesized that a greater percentage of cows would ovulate to hCG resulting in an overall increase in synchronization and conception rates. There was no difference in ovulation rates when comparing the hCG- and GnRH-treated cows (65.7 and 59.6%). However, only cows
16 Template Created By: Damen Peterson 2009 with a follicle greater than or equal to 10 mm on either ovary at the onset of Ovsynch were enrolled into the experiment. This may explain why no difference was observed in ovulation rates as a previous study has reported the tendency of hCG to also ovulate smaller subordinate follicles (Stevenson et al., 2007). Interestingly, Keskin et al. (2010) reported that hCG-treated cows tended to have a decreased synchronization rate when compared to those treated with GnRH (83.8 and 90.1%). Subsequent conception rates at d 30 post-TAI were also significantly decreased in hCG-treated cows compared to those treated with GnRH (37.6 and 48.5%). Follicle size was measured at the time of AI and hCG-treated cows tended to have a smaller dominant follicle than those treated with
GnRH (15.8 ± 0.2 and 16.4 ± 0.2 mm). The authors offer no explanation as to why the synchronization rate differed between treatment groups, but they do suggest that conception rates were possibly affected by dominant follicle size at the time of AI. It has been previously reported that a larger pre-ovulatory follicle at the time of AI has been associated with increased conception rates (Lopes et al., 2007), but this has not always been true (Vasconcelos et al., 1999).
In contrast, other studies incorporating hCG into the basic Ovsynch protocol have reported no reduction in pregnancy rates (De Rensis et al., 2002; De Rensis et al.,
2008a,b). Replacing both injections of GnRH of Ovsynch with hCG in postpartum dairy cows resulted in similar pregnancy rates between hCG-and GnRH-treated cows (39.1 and
37.8%; De Rensis et al., 2002). The final injection of GnRH in the Ovsynch protocol has also been replaced with hCG to evaluate the effectiveness on cows with ovarian cysts (De
Rensis et al., 2008a) and it was reported that cows treated with hCG during the cool time of the year had similar pregnancy rates compared to those treated with GnRH (32 and
17 Template Created By: Damen Peterson 2009
29%). However, cumulative pregnancy rates which included the first TAI and subsequent return service (if necessary) resulted in a significant increase in cumulative pregnancy rate for the hCG treated cows compared to the GnRH group (78 and 56%; De
Rensis et al., 2008a). The authors concluded that replacing the final injection of GnRH with hCG seemed to have a beneficial effect on cumulative pregnancy rates during the cool time of year in cows with ovarian cysts. Similarly, the last injection of GnRH in the
Ovsynch protocol has also been replaced with hCG in cycling cows (De Rensis et al.,
2008b). Cows treated with hCG or GnRH during the summer and winter months did not differ in pregnancy rates, but cumulative pregnancy rates during the summer months were reported to be significantly greater in cows treated with hCG compared to those that received GnRH (63 and 36%). Concentrations of progesterone were also significantly greater from d 3 to 9 post-TAI in cows treated with hCG as compared to those that received GnRH. Embryonic loss was assessed from d 28 to 45 of pregnancy and no difference was indicated between treatments, but it was significantly greater in the summer compared to winter months. It was concluded that hCG improved cumulative pregnancy rates in cows during the summer months when fertility was likely affected by heat stress (De Rensis et al., 2008b).
Presynchronization with hCG has recently been evaluated in beef cows
(Marquezini et al., 2009). Suckled beef cows were subjected to the CO-Synch + CIDR protocol and half received a presynchronization injection of hCG 7 d prior to its onset.
Cows treated with hCG had significantly greater concentrations of progesterone at the onset of the CO-Synch + CIDR protocol than did the cows which did not receive hCG
(1.84 ± 0.19 and 1.22 ± 0.19 ng/mL). The proportion of cows classified as noncycling
18 Template Created By: Damen Peterson 2009 was significantly decreased in those treated with hCG as compared to controls (17 and
37.9%). Pregnancy rates were similar between hCG-treated cows and control cows (54.2 and 53.4%).
For many of these studies, dosage of hCG varied from 500 to 3300 IU (Schmitt et al., 1996; Santos et al., 2001; De Rensis et al., 2002; Stevenson et al., 2007; Keskin et al.,
2010). Recent attempts have been made to determine an adequate dose of hCG that should be administered when used in an estrus synchronization protocol to maximize ovulation (Burns et al., 2008; Buttrey et al., 2010). When 100 µg of GnRH was compared to 500, 1000, 2000, and 3000 IU of hCG in beef cows, there was no difference in ovulation rate between GnRH and any of the hCG treatments as well as no difference between the four doses of hCG (Burns et al., 2008). It was concluded that 500 IU of hCG was sufficient in invoking an ovulatory response similar to GnRH in beef cows. Though, it should be noted that this conclusion was based on a limited number of animals per treatment group (all groups ≤ 11 cows).
Similarly, ovulation rates were compared between dairy cows either administered saline, 100 µg of GnRH, or 500, 1000, 2000, or 3000 IU of hCG (Buttrey et al., 2010). It was reported that hCG ≥ 1000 IU significantly increased ovulation rates over saline but did not differ from GnRH-treated cows, thus suggesting 1000 IU of hCG as an optimal dose equivalent to GnRH. It should be noted that although this particular experiment was limited by number of animals (all groups ≤ 16 cows), a dose response curve was created and the fit was significant. In the same study, a larger sample size (n = 334) of cows were given either saline, 100 µg of GnRH, or 1000 IU of hCG. Results indicated that hCG-treated cows had a significantly greater ovulation rate than those treated with GnRH
19 Template Created By: Damen Peterson 2009 and saline (46.3 > 27.5 > 6.4%). However this difference was only observed in cows with concentrations of progesterone ≥ 1 ng/mL at the time of the injection and not so in cows with concentrations of progesterone < 1 ng/mL. With variable results between studies (De Rensis et al., 2002; Burns et al., 2008; De Rensis et al., 2008a,b; Marquezini et al., 2009; Buttrey et al., 2010; Keskin et al., 2010), more research is needed to better understand hCG’s effect and potential in the synchronization of estrus. In comparison, more consistent results have been achieved by using hCG post-AI to increase conception rates (Santos et al., 2001; Stevenson et al., 2007; Stevenson et al., 2008b; Dahlen et al.,
2010).
The use of hCG Post-AI
It has been reported that cows with decreased concentrations of progesterone as early as 6 d post-AI generally fail to conceive as compared to cows with increased concentrations of progesterone that became pregnant (Mann and Lamming, 1999;
Thatcher et al., 2001; Lopes et al., 2007). To combat this problem, hCG has been used to induce ovulation post-AI to increase endogenous production of progesterone in order to increase conception rates and decrease embryonic loss (Santos et al., 2001; Stevenson et al., 2007; Stevenson et al., 2008b; Dahlen et al., 2010). In one study, hCG and saline were administered 5 d post-AI in high producing dairy cattle to look at the effects on number of CL, plasma progesterone, and conception rates (Santos et al., 2001). Cows in the hCG group had significantly more multiple CL than the control group 14 d post-AI
(86.2 and 23.2%) as well as significantly increased luteal volume. Concentrations of progesterone at 14 d post-AI were 5 ng/mL greater in the hCG-treated cows compared to
20 Template Created By: Damen Peterson 2009 the control cows. Conception rates at 28 d post-AI for the hCG-treated cows were significantly greater than the controls (45.8 and 38.7%, respectively), and remained significantly greater at d 45 and d 90 following AI. Overall, hCG induced an accessory
CL along with increasing concentrations of progesterone and conception rates compared to the control group (Santos et al., 2001).
Similarly, hCG or saline have been administered 7 d post-TAI in suckled beef cows to determine if conception rates were altered at a d 33 diagnosis of pregnancy
(Dahlen et al., 2010). Cows which were later diagnosed as pregnant had significantly greater concentrations of progesterone on d 7 post-TAI as compared to non-pregnant cows (3.7 ± 0.1 and 2.6 ± 0.2 ng/mL). Cows treated with hCG also had significantly greater luteal tissue volume and concentrations of progesterone by d 14 post-TAI than those administered saline. Pregnancy diagnosis at d 33 post-TAI indicated a tendency for increased pregnancy rates in hCG treated cows compared to those that received saline
(56.3 and 50.0%). However, when reevaluated at d 68 for pregnancy, no difference in cumulative pregnancy rate was detected between treatments (Dahlen et al., 2010).
The effects of administering a CIDR, GnRH, or hCG on d 4 to 9 post-TAI in lactating dairy cows has also been investigated (Stevenson et al., 2007). Results were variable among herds but did indicate that hCG significantly increased conception rates in second lactation cows and also overall in two of the five herds used in this study when compared to the untreated control cows. Cows treated with a CIDR tended to increase conception rates but varied among herds while no benefit was observed in GnRH treated cows when compared to controls. Interestingly, more cows were induced to ovulate in both the hCG and GnRH treatmeants but only cows treated with hCG had significantly
21 Template Created By: Damen Peterson 2009 increased concentrations of progesterone 7 d post-treatment. The authors suggest that the reason for variation in results was likely due to the range in days when treatments were administered post-TAI (Stevenson et al., 2007).
In Stevenson et al. (2008b), hCG, GnRH, and saline were administered after the initial diagnosis of pregnancy to determine if hCG or GnRH caused formation of new luteal structures, increased serum concentrations of progesterone, or increased pregnancy survival between d 26 and 71 of pregnancy. The hCG treatment significantly increased the formation of new luteal structures compared to GnRH and saline, while GnRH also significantly increased new luteal structures compared to saline (50 > 26 > 7%). Serum concentrations of progesterone did not differ between treatments, but concentrations for the first two weeks after treatment were significantly increased for cows that formed new luteal structures when compared to those that did not. Pregnancy loss was also not affected by treatments and did not decrease with the formation of additional luteal structures. This, however, is in contrast to a previous study which reported that cows treated with hCG at 33 d post-TAI tended to have increased pregnancy loss compared to those treated with GnRH or untreated controls (Buttrey et al., 2010). It can be concluded that hCG administered between d 5 to 10 post-AI will generally either increase or not change conception rates compared to untreated cows (Santos et al., 2001; Stevenson et al., 2007; Dahlen et al., 2010), while administration of hCG after initial pregnancy diagnosis will not affect or increase pregnancy survival (Stevenson et al., 2008b; Buttrey et al., 2010).
22 Template Created By: Damen Peterson 2009
The use of hCG in Resynchronization Protocols
Another role of hCG that has been investigated is its use in resynchronization protocols. Resynchronization protocols are TAI protocols (typically Ovsynch) that are initiated either prior to (Chebel et al., 2003; Fricke et al., 2003) or after (Pursley et al.,
1997a) pregnancy diagnosis in non-pregnant cows that had been previously inseminated.
The goal of a resynchronization protocol is to improve reproductive efficiency by rapidly reinseminating cows diagnosed as non-pregnant, thereby decreasing days between inseminations and increasing the AI service rate (Fricke, 2002). However, day post-AI of initiation of resynchronization protocols significantly affect conception rates of the subsequent insemination (Fricke et al., 2003).
In one particular study, cows of unknown pregnancy status at d 33 post-AI were administered either 100 µg GnRH, 1000 IU hCG, or served as untreated controls (Buttery et al., 2010). Pregnancy diagnosis was conducted 7 d later and all non-pregnant cows received PGF2α followed 3 d later with GnRH concurrent with TAI (CO-Synch protocol).
There was no difference in conception rates between treatment groups but cows originally diagnosed pregnant that had received hCG but had not been reinseminated, tended to have greater embryonic loss than cows treated with GnRH when reevaluated for pregnancy 4 wk later (6.6 and 3.5%). This is in contrast to a previous study which reported no decrease in pregnancy loss when comparing pregnant cows at d 26 and 71 of pregnancy that had received GnRH, hCG, or saline on d 26 (Stevenson et al., 2008b).
In another study, lactating dairy cows previously inseminated were subjected to one of three resynchronization protocols (Giordano et al., 2010a). The cows in the control group were enrolled into a basic Ovsynch protocol on d 25 post-TAI (Resynch-
23 Template Created By: Damen Peterson 2009
25), and then non-pregnant cows were reinseminated on d 35. The second group consisted of a Double-Ovsynch resynchronization protocol where cows started the protocol on d 22 post-TAI and then those diagnosed non-pregnant were reinseminated on d 49. The final group referred to as HGPG received 2000 IU hCG on d 18 post-TAI and then began the basic Ovsynch protocol on d 25 with non-pregnant cows being reinseminated on d 35 from previous TAI. Double-Ovsynch Resynch and HGPG achieved similar conception rates (37.3 and 35.8%) and both were significantly greater than Resynch-25 (28%). Embryonic loss did not differ among treatment groups when reevaluated at d 53. It was concluded that HGPG offered significantly greater conception rates than Resynch-25 while also decreasing the breeding interval between inseminations by 14 d when compared with the Double-Ovsynch Resynch protocol (Giordano et al.,
2010a).
Similarly, the resynchronization protocols of Resynch-25 and HGPG were compared to GGPG, where GGPG was initiated on d 18 post-TAI with 200 µg of GnRH and the start of the basic Ovsynch protocol on d 25 (Giordano et al., 2010b). Cows receiving HGPG had significantly greater conception rates than those receiving Resynch-
25 (33.0 and 25.3%). In addition conception rates only tended to be greater in the cows receiving GGPG compared to those receiving Resynch-25 (30.8 and 25.3%). However, no statistical difference was detected between the HGPG-and GGPG-treated cows.
Pregnancy loss was assessed at 53 d post-TAI and no differences were reported among treatments. In a subset of cows, synchronization rate was determined by assessing the ovulation rate to the final injection of GnRH and no statistical difference among treatments was indicated. It was concluded that although presynchronization with either
24 Te mplate Created By: Da men Peterson 2009 h C G or G n R H 7 d b ef or e i niti ati o n of R es y n c h- 2 5 di d n ot aff e ct s y n c hr o ni z ati o n r at e, it di d i n cr e as e c o n c e pti o n r at es t o t h e s u bs e q u e nt T AI ( Gi or d a n o et al., 2 0 1 0 b).
T h e us e of h C G i n H eif ers
Over the years, i mprove ments in the synchronization of estrus have brought the d air y i n d ustr y t o t h e p oi nt w h er e eit h er c o m p ar a bl e or gr e at er c o n c e pti o n r at es c a n b e a c hi e v e d t hr o u g h T AI r at h er t h a n d et e ct e d estr us ( El- Z ar k o u n y et al., 2 0 0 4). G e n er all y, inse minating a co w after observed estrus results in conception rates of about 35 %
(Gü me n et al., 2 0 0 3; El- Z ar k o u n y et al., 2 0 0 4), w hil e i n c o m p aris o n, c urr e nt r es e ar c h i n di c at es t h at D o u bl e- O vs y n c h r es ults i n c o n c e pti o n r at es of 4 2 t o 5 0 % ( C u n h a et al.,
2 0 0 8; S o u z a et al., 2 0 0 8; Gi or d a n o et al., 2 0 0 9). H o w e v er, c o m p ar a bl e r es ults i n h eif ers still el u d e d r es e ar c h er t h us f ar. Oft e n, h eif ers d o n ot r es p o n d w ell t o T AI w h e n co mpared to AI fro m detected estrus. Many studies have reported decreases in c o n c e pti o n r at es b y 2 0 t o 4 0 % ( S c h mitt et al., 1 9 9 6; P ursl e y et al., 1 9 9 7 b; St e v e ns o n et al., 2 0 0 0; M arti n e z et al., 2 0 0 2). It h as b e e n s u g g est e d t h at t his dr a m ati c d e cr e as e i n c o n c e pti o n r at es mi g ht b e p artl y a r es ult of h eif ers l a c ki n g a pr o p er o v ul at or y r es p o ns e t o t h e first i nj e cti o n of G n R H i n t h e O vs y n c h pr ot o c ol ( M arti n e z et al., 2 0 0 2). T his c o ul d then lead to pre mature behavioral estrus before the injection of P G F 2 α and thus decrease the efficacy of the subsequent injections of hor mones. Researchers have yet to elucidate why heifers have decreased responsiveness to Gn R H co mpared to dairy co ws but it could b e d u e t o d e cr e as e d d ur ati o n of f olli cl e d o mi n a n c e as s u g g est e d b y At ki ns et al. ( 2 0 0 8) or perhaps reduced release of L H after ad ministration of exogenous Gn R H ( Lucy and
2 5 Template Created By: Damen Peterson 2009
Stevenson, 1986). One potential solution to this problem is the use of hCG in place of
GnRH.
Investigation into administering a single dose of hCG during different stages of the estrous cycle has reported ovulation rates being significantly affected by the stage of the estrous cycle (Price and Webb, 1989). Heifers treated with hCG in the early luteal (d
4 to 7) and late luteal (d 14 to 16) stage of their estrous cycle had greater ovulation rates as compared to those treated with hCG during the mid-luteal stage (d 8 to 13). These results were in agreement with Ireland and Roche (1983) which reported that follicles during the mid-luteal stage were smaller, had fewer granulosa cells, and hCG had a decreased capacity to bind to them due to decreased LH receptors. In another study, hCG was given on d 5 of the estrous cycle and resulted in a greater proportion of hCG-treated dairy heifers to have three follicular waves while untreated control heifers were more likely to have two follicular waves during their respective estrous cycles (Diaz et al.,
1998). In addition to altering follicular dynamics, heifers which ovulated as a result of administration of hCG on d 10 of the estrous cycle, can regress their subsequent CL with
PGF2α by d 2 after ovulation while control heifers first regressed their CL to PGF2α by d 5 after ovulation (Howard and Britt, 1990).
Dahlen et al. (2011) reported that the use of hCG in prepubertal beef heifers caused a significantly greater proportion of follicles to ovulate compared to GnRH (87.5 and 43.8%). Heifers treated with hCG had significantly greater peak concentrations of progesterone 6 d post-treatment compared to the GnRH group (1.72 and 0.31 ng/mL), as well as significantly greater mean volume of luteal tissue (1.54 and 0.23 cm3). Follicular growth was assessed from d 2 to 5 post-treatment and the largest follicle was significantly
26 Template Created By: Damen Peterson 2009 greater in heifers treated with GnRH compared to those treated with hCG. Similarly,
GnRH and hCG have been compared in cycling dairy heifers (Dahlen et al., 2008).
Ovulation rates were significantly greater for hCG-than GnRH-treated heifers (90 and
30%). The largest follicle size measured 4 d post-treatment tended to be greater in the
GnRH than hCG group (11.6 ± 0.7 and 9.6 ± 0.7 mm). Volume of luteal tissue was also examined 7 d post-treatment and was significantly greater in the heifers treated with hCG compared to those treated with GnRH (5.4 ± 0.6 and 3.4 ± 0.6 cm3), but no differences were observed in concentrations of progesterone post-treatment.
Summary
The increased metabolism of steroid hormones such as progesterone and estradiol negatively affect reproductive outcomes in high producing lactating dairy cattle, resulting in decreased conception rates and increased embryonic loss. Estrus synchronization protocols such as Double-Ovsynch not only improves the synchrony of the estrous cycle, but also helps combat the metabolism of steroid hormones by increasing endogenous concentrations of progesterone during follicular growth. It can be concluded that hCG generally has a greater capacity to ovulate follicles in cows and heifers, affect follicular growth, increase the volume of luteal tissue and concentrations of progesterone, and decrease embryonic loss when compared to GnRH. However, with variable results observed in beef and dairy cows and heifers, further research is warranted to determine if hCG offers a viable solution to improving reproductive efficiency in dairy cattle, specifically in the presynchronization stage of the synchronization of estrus.
27 Template Created By: Damen Peterson 2009
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CHAPTER III
MODIFYING THE DOUBLE-OVSYNCH PROTOCOL TO INCLUDE HUMAN
CHORIONIC GONADOTROPIN TO SYNCHRONIZE ESTRUS IN DAIRY
CATTLE
Abstract
The objectives of this study were to determine whether conception, ovulation rates, presynchronization rates, or follicle and CL characteristics were altered after modifying the Double-Ovsynch (DO) protocol to include human chorionic gonadotropin
(hCG) compared to the DO protocol. Protocols were conducted in primiparous and multiparous lactating dairy cows (60 to 458 d postpartum at AI; n = 183), or in nulliparous dairy heifers (13 to16 mo at AI; n = 51). Cows were blocked by parity and randomly assigned to one of two treatments while heifers were stratified by age and breed and randomly assigned to treatments. All females received either an injection of 100 µg of GnRH or 2000 IU of hCG at the initiation of the Pre-Ovsynch (PO) portion of the DO protocol (PO: GnRH/hCG-7d-PGF2α-3d-GnRH). After 7 d, females started the Breeding-
Ovsynch (BO) portion of the DO protocol (BO: GnRH-7d-PGF2α-48/56h-GnRH-16h-
TAI with sex-sorted semen). Transrectal ultrasonography and blood samples were used to assess ovarian structures, ovulation, pregnancy diagnosis (at d 32 or 35 post-TAI), and circulating concentrations of progesterone (P4). Conception rates were similar in females
36 Template Created By: Damen Peterson 2009 treated with GnRH or hCG in cows (32.2 and 25.0%; P > 0.1) and in heifers (30.8 and
36.0%; P > 0.1). Ovulation rates were determined in cows at the onset of PO and were increased with hCG compared to GnRH (77.2 and 62.2%; P < 0.05). Concentrations of
P4 7 d post-hCG or GnRH treatment were greater in cows treated with hCG compared to
GnRH (LSMeans ± SEM; 5.1 ± 0.3 and 3.8 ± 0.4 ng/mL; P < 0.05), but did not differ in heifers (4.5 ± 0.9 and 2.9 ± 0.9 ng/mL; P = 0.14). Luteal regression (P4 < 1.0 ng/mL) from the injection of PGF2α of PO did not differ between GnRH-and-hCG treated cows
(67.0 and 60.9%; P > 0.1) or heifers (42.3 and 56.0%; P > 0.1). Although more cows ovulated to hCG, a greater proportion of these cows tended to fail to have undergone luteolysis by d 3 post-PGF2α compared to cows that had ovulated to GnRH (29.6 and
16.1%; P = 0.09). In contrast, no heifers failed to have undergone luteolysis. The overall percentage of females which were synchronized to PO did not differ between GnRH-or hCG-treated cows (61.5 and 52.2%; P > 0.1) and heifers (42.3 and 40.0%; P > 0.1). In conclusion, no improvement was achieved by replacing the first injection of GnRH in the
DO protocol with hCG.
Key Words: Double-Ovsynch, hCG, presynchronization
Introduction
In the United States, most dairy herds have an estrus detection efficiency of less than 50% (Senger, 1994; Washburn et al., 2002), resulting in significant economic loses for dairy producers (Senger, 1994). But over the past 15 years, estrus synchronization protocols that allow for timed artificial insemination (TAI) have been developed to combat this problem. Their use greatly reduces the need for estrus detection and
37 Template Created By: Damen Peterson 2009 increases the overall reproductive efficiency of the herd (Pursley et al., 1997b). The estrus synchronization protocol referred to as Ovsynch, was introduced to the dairy industry in the mid to late 1990s (Pursley et al., 1995; Pursley et al., 1997a,b). Since then, researchers have developed several modifications to the original Ovsynch.
Conception rates are greatest when the Ovsynch protocol is initiated on d 5 to 12 of the estrous cycle (Vasconcelos et al., 1999; Cartmill et al., 2001). This results in greater rates of ovulation to the first injection of GnRH of Ovsynch and thus increased conception rates (Vasconcelos et al., 1999; Cartmill et al., 2001; Bello et al., 2006). A critical component for successful synchronization of ovulation in dairy cattle involves the inclusion of a presynchronization stage (Moreira et al., 2001; Navanukraw et al., 2004;
Bello et al., 2006). This presynchronization stage increases the likelihood of ovulation to the first injection of GnRH of Ovsynch, leading to increased conception rates
(Vasconcelos et al., 1999; Bello et al., 2006).
Souza et al. (2008) introduced the novel idea of combining two Ovsynch protocols to form what is known as Double-Ovsynch. The first Ovsynch is referred to as
Pre-Ovsynch (PO) and is used for presynchronizing the follicular growth. After the PO, another Ovsynch, the Breeding-Ovsynch (BO), is initiated 7 d later and the cow is inseminated after this Ovsynch. In this study, human chorionic gonadotropin (hCG) was utilized in an attempt to improve the presynchronization stage in both dairy cows and heifers. Human chorionic gonadotropin is a hormone that has similar activity to LH
(Farin et al., 1988), inducing ovulation by binding to LH receptors on the follicle
(Schmitt et al., 1996a; Stevenson et al., 2007) and also producing LH-like effects (Farin et al., 1988). Therefore, ovulation of a follicle using hCG is no longer dependent upon
38 Template Created By: Damen Peterson 2009 the LH surge produced from an injection of GnRH (Kinser et al., 1983). Research has indicated that hCG has an increased capacity to induce ovulation when compared to
GnRH (Stevenson et al., 2007; Buttrey et al., 2010) although not so in all (Burns et al.,
2008; Keskin et al., 2010). Heifers submitted to the Ovsynch protocol have significantly decreased conception rates as compared to breeding to detected estrus (Pursley et al.,
1997a). It is thought that this decrease is predominantly caused by the failure of ovulation after the initial injection of GnRH of Ovsynch (Moreira et al., 2000; Martínez et al., 2002). However, induction of ovulation with hCG in dairy heifers has been reported to be significantly increased compared to GnRH (Dahlen et al., 2008).
Therefore, we hypothesized that replacing the first injection of GnRH in the
Double-Ovsynch protocol with hCG would increase the percentage of females that ovulate, thus improving the overall presynchronization rate leading to increased conception rates. The objectives of this study were to determine whether conception, ovulation rates, presynchronization rates, or follicle and CL characteristics were altered after modifying the Double-Ovsynch protocol to include hCG compared to the Double-
Ovsynch protocol.
Materials and Methods
Herd Management Practices
All procedures in this study were approved by the Institutional Animal Care and
Use Committee of Mississippi State University. Experiment 1 was conducted in lactating dairy cows while Experiment 2 was conducted in dairy heifers. Cows were housed in
39 Template Created By: Damen Peterson 2009 two free-stall barns at the Bearden Dairy Research Center, Mississippi State, MS. Cows had access to water ad libitum and were fed twice daily with a total mixed ration consisting of corn silage, rye grass bailage, dry bermuda hay, cottonseed, and a complete grain mix. Rations were formulated to meet or exceed dietary requirements of lactating dairy cows (NRC, 2001). Pens were equipped with feedline head lockups and free stalls were bedded with sand. Cows were milked twice daily and received recombinant bovine somatotropin at 90 DIM and every 14 d thereafter. Cows in Experiment 1 consisted of
Holstein (n = 146) and Jersey (n = 37) cows. Due to negative effects of heat stress in
Mississippi during summer months, cows in this herd were seasonally bred with no cows being inseminated from the end of May to mid November.
In Experiment 2, all breeding eligible dairy heifers were taken off pasture during the fall 2009 breeding season and housed in a separate free stall barn at the Bearden
Dairy Research Center. Holstein (n = 41) and Jersey (n = 10) heifers ranged in age from
13 to 16 mo at the time of AI. Range in age for first AI was due to all females being seasonally born as previously described. Heifers had free access to water and stalls were bedded with sand. For all procedures, heifers were restrained in feedline head lockups.
Heifers were fed once daily with a diet comprised of corn silage and feed refusals from the lactating dairy cows. Once diagnosed pregnant, heifers were returned to the pasture.
Experimental Design and Treatments
Protocols in Experiment 1 were conducted in primiparous (n = 67) and multiparous (n = 116) dairy cows during the fall 2009 and 2010 breeding seasons. Cows were grouped into 3 replicates in both yr 1 and 2 with enrollment beginning in late
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October and ending in early December. The first group enrolled each yr consisted of hold-over cows (239 to 458 DIM at TAI; n = 64) from the previous breeding season that had failed to become pregnant. Groups 2 and 3 of both yr then consisted of early lactation cows (60 to 116 DIM at TAI; n = 119) receiving their first TAI post-partum.
Cows were blocked by parity and randomly assigned to 1 of 2 treatments (Figure 1).
Before cows received treatments, treatment groups were checked for balance in milk production and DIM as to not cause any bias with results. Likewise, heifers in
Experiment 2 were grouped into 2 replicates with enrollment of both groups in early
November. Heifers were stratified by age and breed and then randomly assigned to 1 of
2 treatments. All females then received either an injection of 100 µg (2 mL) of GnRH i.m. (Cystorelin; Merial Ltd., Duluth, GA) or 2000 IU (2 mL) of hCG i.m. (Chorulon;
Intervet Inc., Millsboro, DE) at the initiation of the PO portion of the Double-Ovsynch protocol (d 0). Seven days later, all females received 25 mg (5 mL) PGF2α i.m. (Lutalyse;
Pfizer Animal Health, New York, NY) followed 3 d later (d 10) with an injection of
GnRH (PO: hCG/GnRH-7 d- PGF2α-3 d-GnRH). After 7 d, females started the BO
portion of the Double-Ovsynch protocol (BO: GnRH-7d-PGF2α-48/56h-GnRH-16h-TAI).
All females received TAI with sex-sorted (female) semen chosen by the herd manager. A
total of 18 sires were used between treatments in dairy cows and 9 sires were used
between treatments in heifers. For the dairy cows, inseminations were performed by 3
technicians and were randomly assigned, while a single technician performed
inseminations in all heifers.
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Milk Production and BCS
For yr 1 and 2 of both experiments, BCS of all females were taken by a single trained observer at the time of the first injection of hCG or GnRH (d 0). For yr 2, BCS in cows were also taken at the time of AI and at pregnancy diagnosis on d 32 post-TAI.
Scores were based on a 5 point scale: 1 = severe underconditioning to 5 = severe overconditioning (Edmonson et al., 1989). Milk production was recorded monthly on individual cows and the test-day milk yield nearest to the initiation of the trial was used to assess its effect on concentrations of progesterone (P4) in plasma, ovarian characteristics, and ovulation and conception rates.
Ovarian Measurements and Pregnancy Diagnosis
Transrectal ultrasonography (yr 1: 5.0-MHz linear-array transducer, Aloka 500V,
Corometrics Medical Systems Inc., Wallingford, CT; yr 2: 10.0 to 5.0-MHz linear-array transducer, MicroMaxx, SonoSite, Inc., Bothell, WA) was conducted for examination of ovarian structures on cows on d 0, 7, and 10 while only on d 7 and 10 in heifers (Figure
1). Diameters were recorded of all corpora lutea (CL) along with all follicles ≥4 mm.
Ovulation from the injection of hCG or GnRH on d 0 was confirmed when a newly formed CL on d 7 was detected on the ovary where a follicle had previously been located.
Ovaries of heifers were not examined on d 0 to determine ovulation rates but a blood sample was taken to assess concentrations of P4. Volume of luteal tissue was determined by using the formula: [4/3 x R3 x π]. The radius (R) of the luteal structure was calculated
by using the formula: R = (W/2 + L/2)/2, where W = width and L = length of the luteal
structure. In CL with a fluid-filled cavity, volume of the cavity was calculated and
42 Template Created By: Damen Peterson 2009 subtracted from the total volume of the CL. Pregnancy diagnosis was performed on d 32
(cow) or 35 (heifer) post-TAI by transrectal ultrasonography and was confirmed by palpation per rectum between d 60 and 90. Non-pregnant cows were resynchronized with
Ovsynch on d 32 post-TAI during yr 1. In yr 2, cows were resynchronized with Ovsynch on d 25 post-TAI and all non-pregnant cows were reinseminated.
Blood Collection and RIA
Blood samples were taken on d 0, 7, 10, and 17 (Figure 1) from all females via the median coccygeal vein just prior to injection of hormones. Samples were collected in evacuated tubes containing the anticoagulant K2 EDTA (Becton Dickinson, Franklin
Lakes, NJ) and immediately placed on ice. Within 3 h of collection, samples were transported to the laboratory and centrifuged at 3,000 g at 4°C for 20 min. Plasma was collected and stored at -20°C for later assessment of concentrations of P4 using solid- phase competitive binding RIA kits (Coat-A-Count, Siemens Medical Solutions
Diagnostics, Los Angeles, CA). Inter- and Intra-assay coefficients of variance were 6.2 and 2.2%, respectively. Females with concentrations of P4 ≥ 1.0 ng/mL on d 0 were classified as having increased concentrations of P4 at the beginning of PO while females with P4 < 1.0 ng/mL were classified as having decreased concentrations of P4. Luteal regression from d 7 to 10 was defined as regression of the CL from the injection of PGF2α
(d 7 P4 ≥ 1.0 ng/mL and d 10 P4 < 1.0 ng/mL). Females undergoing early luteal regression were defined as not ovulating and having P4 ≥ 1.0 ng/mL on d 0 and then P4 <
1.0 ng/mL on d 7. Synchronization rate to the PO was defined as luteal regression from
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the injection of PGF2α by d 10 and then the presence of a functional CL (P4 ≥ 1.0 ng/mL) on d 17.
Statistical Analyses
The GLIMMIX procedure of SAS (SAS Inst. Inc., Cary, NC) was used to analyze all binomial distributed data (i.e., increased P4 on d 0 and 17, ovulation, luteal regression, synchronization, and conception rates), and continuous data (i.e., concentrations of P4, diameter of the largest follicle located on either ovary on d 0, 7, and 10 as well as number and volume of CL on d 0 and 7). A separate analysis was also conducted to determine differences in ovarian characteristics and concentrations of P4 in cows that ovulated from either the injection of hCG or GnRH. Cows and heifers were separated for the analysis of data. Means are presented as a percentage for binomial distributed data while least square means are given for all ovarian and measurements of P4. Means and least square means were separated when a P-value of ≤ 0.1 was detected. Standard errors of means are presented. For cows, variables considered for the statistical model were treatment, increased P4 on d 0 or 17, breed, parity, group, BCS (categorized as decreased [BCS ≤
2.5] or moderate [BCS ≥ 2.75] body conditioning), AI technician, sire, and yr. Milk production and DIM were included as covariates. For heifers, variables considered for the statistical model were treatment, increased P4 on d 7 and 17, breed, group, BCS
(categorized as decreased [BCS ≤2.75] or moderate [BCS ≥ 3.0] body conditioning), age
(categorized as < 15 or ≥ 15 mo of age at AI; median = 15 mo), and sire. Variables were removed or retained from the statistical models in a backward stepwise procedure using a
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Wald statistic of P > 0.10. All two-way interactions between variables were considered for the analysis of data.
Results
Experiment 1: Dairy Cows
Ovarian Responses to hCG or GnRH during Pre-Ovsynch
Ovulation rates at the onset of PO were increased (P < 0.05) in cows treated with hCG compared to GnRH (77.2 and 62.2%, respectively). No interaction was detected between treatment and P4 status (≥ 1 or < 1 ng/mL) at the onset of PO (P = 0.13) on ovulation rates. However, cows with increased concentrations of P4 (≥ 1 ng/mL) on d 0 had greater (P = 0.01) ovulation rates when treated with hCG as compared to GnRH
(79.3%, n = 58; and 54.9%, n = 51). Concentrations of P4 in plasma on d 0 did not differ
(P > 0.1) between cows treated with hCG or GnRH (LSMeans ± SEM; 3.5 ± 0.4 and 2.9
± 0.4 ng/mL). The percentage of cows with increased concentrations of P4 on d 0 was similar (P > 0.1) between cows treated with hCG or GnRH (63.0 and 57.1%). Number of
CL on d 0 located on both ovaries tended (P = 0.1) to be greater in cows treated with hCG than GnRH (1.2 ± 0.1 and 1.0 ± 0.1). However, total CL volume did not differ (P >
0.1) between treatments (Table 1). Diameter of the largest follicle located on either ovary was similar (P > 0.1) between cows treated with hCG or GnRH (12.9 ± 0.5 and 13.4 ±
0.5 mm) but multiparous cows had greater (P < 0.01) follicle diameter than primiparous cows (14.3 ± 0.5 and 12.0 ± 0.6 mm; Table 5). For cows that ovulated, diameter of the
45 Template Created By: Damen Peterson 2009 largest follicle located on either ovary on d 0 did not differ (P > 0.1) between cows treated with hCG or GnRH (13.5 ± 0.6 and 14.1 ± 0.7 mm; Table 2). Multiparous cows that ovulated had similar (P > 0.1) follicle diameter as primiparous cows (15.1 ± 0.5 and
14.3 ± 0.6 mm). In cows that ovulated, concentrations of P4 on d 0 did not differ (P =
0.13) between cows treated with hCG or GnRH (2.3 ± 0.3 and 1.6 ± 0.3 ng/mL). Number of CL on d 0 located on both ovaries were greater (P < 0.05) in cows that ovulated to hCG as compared to GnRH (1.2 ± 0.1 and 0.9 ± 0.1) but volume of luteal tissue did not differ (P > 0.1; Table 2).
Measurements at time of PGF2α of Pre-Ovsynch
Concentrations of P4 on d 7 were greater (P < 0.01) in all cows treated with hCG compared to GnRH (4.3 ± 0.3 and 3.0 ± 0.3 ng/mL; Table 1) as well as in only cows that ovulated in response to hCG or GnRH (5.1 ± 0.3 and 3.8 ± 0.4 ng/mL; Table 2). For all cows, the percentage with increased concentrations of P4 on d 7 tended (P = 0.1) to be greater in hCG-treated cows than those treated with GnRH (83.7 and 73.6%). Number of
CL on d 7 located on both ovaries was greater (P < 0.001) in all cows treated with hCG than with GnRH (1.9 ± 0.1 and 1.3 ± 0.1, respectively). Total volume of CL tissue was also greater (P < 0.01) in hCG-treated cows over those treated with GnRH (7.5 ± 0.6 and
5.1 ± 0.6 cm3). Similar results of significantly increased number and volume of CL on d
7 was also observed for cows that had ovulated to the injection of hCG compared to
GnRH (Table 2). In all cows, diameter of the largest follicle located on either ovary on d
7 tended (P = 0.08) to be larger in cows treated with GnRH compared to those treated with hCG (13.7 ± 0.4 and 12.7 ± 0.4 mm). When compared to just cows that ovulated,
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GnRH treated cows had greater (P < 0.05) follicle diameter on d 7 when compared to hCG (13.8 ± 0.5 and 12.4 ± 0.5 mm). Multiparous cows again had increased (P = 0.05) follicle diameter on d 7 over primiparous cows (13.8 ± 0.5 and 12.6 ± 0.5 mm; Table 5).
Responses to PGF2α and Day 10 Measurements
Unlike on d 7, diameter of the largest follicle on either ovary on d 10 did not differ (P > 0.1) between cows treated with hCG or GnRH (14.4 ± 0.4 and 14.9 ± 0.4 mm) and no difference (P > 0.1) was observed in cows that had ovulated to either hCG or
GnRH (14.8 ± 0.4 and 15.6 ± 0.5 mm). Follicle diameters in multiparous cows differed
(P < 0.05) from that observed in primiparous cows (15.3 ± 0.4 and 14.0 ± 0.5 mm).
Concentrations of P4 on d 10 were greater (P < 0.01) in cows treated with hCG than those treated with GnRH (1.1 ± 0.1 and 0.5 ± 0.1 ng/mL, respectively). Concentrations of P4 on d 10 were affected (P = 0.07) by an interaction of treatment by parity in all cows and in those that ovulated after treatments on d 0 (P = 0.05; Figure 2). The percentage of cows with decreased concentrations of P4 (< 1 ng/mL) by d 10 tended (P = 0.07) to be greater in cows treated with GnRH compared to hCG (81.3 and 69.6%). When comparing only cows that ovulated after d 0, those treated with hCG had greater (P =
0.001) concentrations of P4 on d 10 than those treated with GnRH (1.2 ± 0.2 and 0.4 ±
0.2 ng/mL; Table 2). Likewise, in cows that ovulated, the percentage with decreased concentrations of P4 by d 10 was greater (P < 0.05) in cows treated with GnRH than hCG
(84.2 and 66.2%).
Overall, luteal regression by d 10 from the injection of PGF2α of PO did not differ
(P > 0.1) between GnRH-and hCG-treated cows (67.0 and 60.9%; Table 3) and no
47 Template Created By: Damen Peterson 2009 difference (P > 0.1) was observed in the percentage of cows undergoing early luteal regression between hCG and GnRH treated cows (15.2 and 19.8%). No treatment by
BCS status interaction was detected (P = 0.33) for luteal regression, but cows treated with
GnRH and having decreased body conditioning (BCS < 2.75) had greater (P = 0.04) luteal regression than those treated with hCG and having moderate (BCS ≥ 2.75) body conditioning (73.9%, n = 23; 61.2%, n = 67). Eight cows ranging in P4 from 0.75 to 0.95 ng/mL on d 7 were considered to have undergone luteal regression based on ovulation from d 0 to 7 and P4 < 0.1 ng/mL on d 10 (GnRH, n = 5; hCG, n = 3). Three cows ranging in P4 from 1.0 to 1.4 ng/mL on d 10 were considered to be undergoing luteal regression based on a decrease in P4 from d 7 of greater than 75%. Two cows ranging in
P4 from 1.1 to 1.2 ng/mL were also considered to be undergoing early luteal regression based on no detection of ovulation and a greater than 75% decrease in P4 from d 0 to 7.
The proportion of cows failing to undergo luteal regression by d 10 was greater (P <
0.05) in hCG than GnRH-treated cows (23.9 and 12.1%). However, of the cows that ovulated, the proportion failing to undergo luteal regression by d 10 tended (P = 0.09) to differ in cows treated with hCG than those treated with GnRH (29.6 and 16.1%). The percentage of cows synchronized to PO did not differ (P > 0.1) between cows treated with hCG or GnRH (52.2 and 61.5%). Synchronization rate, however, was greater (P <
0.05) in cows that had been treated with GnRH on d 0 and ovulated compared to those that ovulated to hCG (80.4 and 60.6%). Synchronization rate tended (P = 0.09) to be affected by DIM, with hold-over cows having the greatest proportion synchronized as compared to those in early lactation (65.6 and 52.9%). Although no interaction was detected between treatment and parity, primiparous cows treated with GnRH tended (P =
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0.06) to have a greater proportion synchronized to PO as compared to those that were treated with hCG (65.7%, n = 35; and 43.8%, n = 32).
Evaluation of Breeding-Ovsynch
The proportion of cows with increased concentrations of P4 on d 17 (onset of BO) was similar (P > 0.1) between cows treated with hCG or GnRH (82.6 and 86.8%).
Concentrations of P4 on d 17 were less (P < 0.05) in cows originally treated with hCG as compared to GnRH (2.8 ± 0.2 and 3.5 ± 0.2 ng/mL). When evaluated between cows synchronized to PO, no difference (P > 0.1) was detected between treatments of hCG or
GnRH (3.2 ± 0.2 and 3.5 ± 0.2 ng/mL). Multiparous cows tended (P = 0.08) to have greater P4 on d 17 as compared to primiparous cows (3.6 ± 0.2 and 2.9 ± 0.3; Table 5), with multiparous cows treated with GnRH on d 0 having greater (P = 0.05) concentrations of P4 than primiparous cows treated with hCG (3.9 ± 0.4 ng/mL, n = 56 and 2.6 ± 0.5 ng/mL, n = 32). No difference (P = 0.11) in concentrations of P4 was observed between primiparous and multiparous cows synchronized to PO (3.1 ± 0.2 and
3.6 ± 0.2 ng/mL). Conception rates evaluated on d 32 post-TAI did not differ (P > 0.1;
Table 4) between cows presynchronized with hCG compared to GnRH (25.0 and 32.2%).
Cows with increased concentrations of P4 on d 17 had similar (P = 0.85) conception rates from that of cows with decreased concentrations of P4 (29.9%, n = 154; and 21.4%, n =
28). Conception rates also did not differ (P = 0.28) between cows synchronized to PO as compared to those that were not (31.4%, n = 105; and 24.7%, n = 77). Although no effect of AI technician was noted on conception rates (P = 0.37), an interaction of treatment by technician was observed (P < 0.05; Figure 3).
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Experiment 2: Dairy Heifers
Ovarian Responses to hCG or GnRH during Pre-Ovsynch
No difference (P > 0.1) was observed in concentrations of P4 on d 0 between heifers that received hCG or GnRH (3.3 ± 0.6 and 2.4 ± 0.6 ng/mL; Table 6). The percentage of heifers with increased concentrations of P4 on d 0 was similar (P > 0.1) between those treated with hCG or GnRH (56.0 and 57.7%). Concentrations of P4 and
P4 status on d 0 were, however, affected by age and breed of heifer (Table 8). Heifers younger than the median age of 15 mo at AI had decreased (P < 0.05) concentrations of
P4 on d 0 when compared to those that were older (2.0 ± 0.6 and 3.7 ± 0.6 ng/mL), and the proportion of heifers with increased P4 on d 0 was also lesser (P < 0.01) in younger heifers than those that were older (38.5 and 76.0%). No difference (P = 0.13) in concentrations of P4 on d 0 was observed between Holstein and Jersey heifers (2.5 ± 0.5 and 4.2 ± 0.9 ng/mL) but a greater (P < 0.05) proportion of Jersey heifers had increased
P4 over Holstein heifers (80.0 and 51.2%).
On d 7, concentrations of P4 did not differ (P = 0.14) between hCG-and GnRH- treated heifers (4.5 ± 0.9 and 2.9 ± 0.9 ng/mL) and no difference (P = 0.20) was observed in the proportion of heifers with increased concentrations of P4 between treatments of hCG and GnRH (56.0 and 38.5%). Again, age and breed of heifer on d 7 affected the concentrations of P4 along with the proportion of heifers with increased P4 (Table 8).
Heifers were then classified by whether or not they had decreased (< 1 ng/mL) concentrations of P4 on both d 0 and 7 of PO (Low-Low). The percentage of heifers classified as Low-Low did not differ (P > 0.1) between treatments of hCG and GnRH
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(20.0 and 26.9%) but was affected by age and breed of the heifer. More heifers less than the median age of 15 mo at AI tended (P = 0.07) to be classified as Low-Low as compared to those that were older (34.6 and 12.0%), while breed also tended (P = 0.06) to affect the proportion of heifers classified as Low-Low (Holstein, 29.2%; and Jersey,
0%).
Number of CL on both ovaries on d 7 was greater (P < 0.05) in heifers treated with hCG compared to GnRH (1.5 ± 0.2 and 0.9 ± 0.2) and heifers treated with hCG tended (P = 0.08) to have greater CL volume than those treated with GnRH (4.5 ± 0.8 and
2.5 ± 0.8 cm3). Number of CL on d 7 tended (P = 0.07) to be affected by breed of the heifer, while CL volume was affected (P < 0.05) by age (Table 8). Interestingly, 41.7%
(5/12) of heifers classified as Low-Low had an identifiable luteal structure on d 7 of PO.
Concentrations of P4 on d 0 for these 5 heifers were all < 0.1 ng/mL while P4 on d 7 ranged from 0.4 to 0.6 ng/mL. Heifers treated with hCG had greater (P = 0.05) follicle diameter than those treated with GnRH (12.8 ± 0.7 and 10.9 ± 0.8 mm) on d 7 of PO. An interaction of treatment by P4 status (< 1 or ≥ 1 ng/mL) tended (P = 0.08) to affect follicle diameter on d 7 (Figure 4). Follicle size was not affected by age, but tended to be affected by breed (P = 0.10; Table 8).
Responses to PGF2α and Day 10 Measurements
Concentrations of P4 on d 10 tended (P = 0.1) to be greater in heifers treated with hCG compared to GnRH (0.4 ± 0.1 and 0.2 ± 0.1 ng/mL), however the percentage of heifers with decreased P4 by d 10 did not differ (P > 0.1) between treatments (hCG,
96.0%; and GnRH, 96.2%). Luteal regression from the injection of PGF2α was similar (P
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> 0.1) between hCG and GnRH treated heifers (56.0 and 38.5%; Table 7), with no difference (P > 0.1) also observed in the percentage of heifers undergoing early luteal regression (hCG, 24.0%; and GnRH, 30.8%). Luteal regression was not affected (P =
0.11) by age but tended (P = 0.07) to be affected by breed (Table 8). The diameter of the largest follicle located on either ovary on d 10 was not analyzed for heifers due to a large proportion of heifers already ovulating by that time. However, the percentage of heifers ovulating by d 10 of PO was similar (P > 0.1) between hCG and GnRH treatments (47.8 and 47.6%). Heifers synchronized to PO did not differ (P > 0.1) between hCG and
GnRH treatments (40.0 and 42.3%) but was affected (P < 0.01) by age of heifer (< 15 mo, 23.1%; and ≥ 15 mo, 60.0%).
Evaluation of Breeding-Ovsynch
Concentrations of P4 on d 17 were similar (P > 0.1) between hCG-and GnRH- treated heifers (3.9 ± 0.5 and 4.7 ± 0.5 ng/mL), with no difference (P > 0.1) observed in the proportion of heifers with increased P4 (hCG, 80.0%; and GnRH, 88.5%). Older heifers (≥ 15 mo of age at TAI) had greater (P = 0.05) concentrations of P4 on d 17 than those that were younger (5.0 ± 0.5 and 3.5 ± 0.5 ng/mL) as well as a greater (P < 0.05) percentage of older heifers with increased P4 over those that were younger (100 and
69.2%). Conception rates did not differ (P > 0.1) between heifers presynchronized with hCG compared to GnRH (36.0 and 30.8%). Heifers synchronized to PO had similar (P =
0.59) conception rates as compared to those not synchronized (42.9%, n = 21; and 26.7%, n = 30). However, conception rates were greater (P < 0.05) in heifers that were older as compared to those that were younger (48.0 and 19.2%).
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Discussion
The main goal of the current study was to increase the ovulation rate to the first injection of Double-Ovsynch by replacing GnRH with hCG and to determine whether this increase in ovulation rate would lead to a greater proportion of females becoming synchronized to PO and improve overall conception rates. Previous research has indicated that increasing ovulation rates to the first injection of GnRH of Ovsynch results in an increased proportion of cows regressing their CL due to the administration of exogenous PGF2α (Bello et al., 2006) and also ovulating to the final injection of GnRH of
Ovsynch (Bello et al., 2006; Rutigliano et al., 2008). Our findings indicated that hCG induced more cows to ovulate when compared to GnRH. This is in agreement with some
(Stevenson et al., 2007; Stevenson et al., 2008; Buttrey et al., 2010) but not all (Schmitt et al., 1996a; Burns et al., 2008; Keskin et al., 2010) studies evaluating hCG and GnRH. In agreement with our results, it has been reported that hCG causes improved ovulation rates over GnRH in cows with increased P4 (Buttrey et al., 2010). This is not surprising due to recent research indicating that increasing the dose of GnRH from 100 to 200 µg when administered on d 7 of the estrous cycle (increased P4 status) significantly increased ovulation rates (Giordano et al., 2009). Although it should be noted that luteal regression and conception rates did not differ in this study (Giordano et al., 2009). Interestingly, reducing both injections of GnRH of Ovsynch from 100 to 50 µg when initiated on a random day of the estrous cycle had similar synchronization and conception rates (Fricke et al., 1998). However, ovulation rates to the first GnRH of Ovsynch and concentrations of P4 were not assessed in this particular study (Fricke et al., 1998). The increased ovulation rates observed in cows treated with hCG can likely be attributed to the ability
53 Template Created By: Damen Peterson 2009 of hCG to act independent of the pituitary gland (De Rensis et al., 2010) and by its longer half-life (Wehmann and Nisula, 1981). It has been reported that heifers treated with hCG had significantly increased circulating concentrations of hCG 30 h post-treatment and had still not returned to basal concentrations by 66 h post-treatment (Schmitt et al., 1996a).
In comparison, the LH-induced surge from the injection of GnRH lasts about 5 h until returning to basal concentrations (Chenault et al., 1990). The longer exposure of hCG binding to the LH receptors could then result in greater ovulation rates (Buttrey et al.,
2010).
As previously reported, the volume of luteal tissue and the number of CL were significantly increased in cows and heifers that received hCG as compared to GnRH
(Schmitt et al., 1996b; Stevenson et al., 2007; Stevenson et al., 2008). Increase in volume of luteal tissue has been associated with increased concentrations of P4 (Vasconcelos et al., 2001; Stevenson et al., 2007; Stevenson, 2011). In agreement with previous literature, our results also indicated that cows treated with hCG had significantly increased volume of luteal tissue as well as concentrations of P4 when compared to cows administered GnRH. This could be due to the ability of hCG to increase concentrations of
P4 on the existing CL by causing hypertrophy of luteal cells (Fricke et al., 1993; Schmitt et al., 1996b; Stevenson et al., 2007) and also by increasing the formation of accessory
CL (Schmitt et al., 1996a; Santos et al., 2001; Galvão et al., 2006; Stevenson et al.,
2007). In the ewe, administration of hCG on d 5 of the estrous cycle significantly increases the proportion of large luteal cells and subsequently reduce the proportion of small luteal cells (Farin et al., 1988). This should then increase endogenous
54 Template Created By: Damen Peterson 2009 concentrations of P4 due to large luteal cells typically secreting 2 to 40 times more P4 than small luteal cells (Niswender et al., 2000).
Concentrations of P4 in the current study were only greater in cows on d 7 of PO that received hCG and not in heifers. This could be attributed to a large proportion of heifers being classified as Low-Low and several of them having a nonfunctional CL on d
7 of PO. Cycling status was not assessed prior to the onset of the trial in heifers or cows, but it is likely that many of the heifers younger than the median age of 15 mo at AI were pre-pubertal and thus noncycling at the onset of PO. Throughout the majority of
Experiment 2, parameters measured to assess reproductive outcomes were affected by both age and breed of heifer. Dairy heifers generally reach puberty around 9 to 11 mo of age when they are at approximately 40% of their mature body weight (Sejrsen, 1994).
However bodyweights were not taken for heifers in the current study. Obtainment of puberty can be affected by several factors such as nutrition (Schillo et al., 1992), breed
(Stewart et al., 1980; Getzewich, 2005), genetics (Stewart et al., 1980), or season (Menge et al., 1960; Schillo et al., 1992). In Experiment 2, all heifers were ≥ 12 mo of age at the onset of PO. Seven Jersey heifers were classified as younger than the median age but none were classified as Low-Low. This is not surprising since obtainment of puberty has been reported to be at an earlier age in Jersey than Holstein heifers (Getzewich, 2005;
Williams, 2007). With a large proportion of heifers not having increased concentrations of P4 on d 7 (≥ 1 ng/mL), it is difficult to determine if this was a result of failure of ovulation from treatments, heifers being pre-pubertal before treatments, or possibly a combination of both.
55 Template Created By: Damen Peterson 2009
It was unforeseen that a greater proportion of cows in Experiment 1 ovulating to hCG would then fail to undergo successful luteal regression (< 1 ng/mL) by d 3 post-
PGF2α as compared to those that received GnRH. This was true when analyzing all cows and when analyzing only those that had originally ovulated to hCG. In particular, failure of luteal regression by d 10 was most evident in primiparous cows that had received hCG on d 0. These cows had significantly greater concentrations of P4 on d 10 and the greatest failure of luteal regression compared to all others. To the knowledge of the authors, only one previous study has reported a negative effect of hCG on subsequent luteal regression (Shipley et al., 1988). In this particular study, dairy heifers were synchronized with PGF2α and administered 2500 IU of hCG 48 h post-estrus and then
PGF2α 5 to 9 d later. Overall luteal regression rates were significantly reduced in heifers treated with hCG as compared to untreated control heifers. Interestingly, luteal regression did not differ on d 6 of the estrous cycle but was significantly reduced by d 8 in heifers treated with hCG as compared to control heifers (Shipley et al., 1988). In contrast, Bennett et al. (1991) reported no difference in luteal regression between hCG and untreated control heifers on d 7 and 13 of the estrous cycle when 2500 IU of hCG had been originally administered 48 h post-estrus. Surprisingly, in contrast to Shipley et al. (1988), no differences in concentrations of P4 were detected on either d 7 or 13 between the control heifers and heifers treated with hCG (Bennett et al., 1991).
Moreover, no heifers in Experiment 2 of the current study were classified as having failed luteal regression. Previous research has also reported that when hCG was administered to dairy heifers on d 10 of the estrous cycle to induce an accessory CL, both original and induced CL underwent luteal regression when administered PGF2α on either d 2, 4, or 6
56 Template Created By: Damen Peterson 2009 after ovulation (Howard and Britt, 1990). In dairy cows, De Rensis et al. (1999) reported no difference in time to estrus after administering PGF2α on d 6 or 9 after initial treatment of hCG (2000 IU) or GnRH on d 0 (random day of the estrous cycle), thus suggesting no difference in luteal regression.
Failure of luteal regression can be attributed to several factors. One possibility is that the cow may be in metestrus (d 1 to 4 of estrous cycle) at the time of the injection of
PGF2α. After ovulation, a newly formed CL has been found to be unresponsive to PGF2α treatment before d 5 of the estrous cycle in cattle (Tsai and Wiltbank, 1998; Acosta et al.,
2002). It is unlikely that failure of luteal regression was affected by cows in metestrus in the present study. The criteria used to determine luteal regression has traditionally been defined as cows needing to have concentrations of P4 ≥ 1 ng/mL on the day of the injection of PGF2α and thus be classified as having a functional CL and no longer in metestrus (El-Zarkouny et al., 2004; Bello et al., 2006; Santos et al., 2010). In the present study, only one cow with P4 ≥ 1 ng/mL on d 7 of PO increased P4 by d 10.
Another factor that could affect luteal regression is decreased PGF2α receptors on the CL. When hCG was administered on d 2 of the estrous cycle in dairy heifers, it was reported that there was a significant decrease in large luteal cells and subsequent binding of PGF2α on d 7 as compared to untreated control animals (Bennett et al., 1989).
However, no difference in the proportion of large luteal cells or binding of PGF2α was detected later on either d 11 or 13 of the estrous cycle. The decrease in large luteal cells is critical to luteal regression due to the majority of PGF2α receptors being located on large luteal cells (Chegini et al., 1991). In contrast, administration of hCG on d 5 of the estrous cycle in the ewe has been reported to increase the proportion of large luteal cells
57 Template Created By: Damen Peterson 2009 in the CL by d 10 (Farin et al., 1988). It was proposed by Niswender et al. (1985) that stem cells located in the newly forming CL of the ewe could differentiate into small luteal cells, which could then also differentiate into large luteal cells under physiological concentrations of LH. Perhaps failure of luteal regression in the current study was partly a result of hCG delaying the natural mechanism of converting small luteal cells into large luteal cells and thus decreasing the binding capacity of PGF2α as suggested by Bennett et al. (1989) for cows that were in early metestrus on d 0 of PO. For cows ovulating to hCG, perhaps the prolonged half-life of hCG can also affect subsequent early luteal formation and increase the failure of luteal regression. However, it has also been reported that LH administered at physiological concentrations in the ewe does not increase the proportion of small to large luteal cells (Farin et al., 1990).
A possible solution to decreasing the failure of luteal regression in the current study could be remedied by administering two injections of PGF2α. When a single dose of PGF2α (500 µg cloprostenol) was administered in a 5-d CO-Synch protocol in dairy cows, approximately half of the cows that ovulated to the initial injection of GnRH underwent luteal regression by 72 h (Santos et al., 2010). However, luteal regression was significantly increased to 96% when PGF2α was given on both d 5 and 6 post-GnRH.
This was to be expected because many of the cows ovulating after the initial GnRH would likely still be in metestrus and thus unresponsive to PGF2α by d 5. Interestingly, a third group of cows in this study received the standard CO-Synch-72 protocol, and those ovulating to the initial injection of GnRH only had a luteal regression rate of 71.4%
(Santos et al., 2010). Similarly, a significant increase in the number of animals that underwent luteal regression was also reported when two doses of PGF2α (25 mg
58 Template Created By: Damen Peterson 2009 dinoprost) were given 24 h apart in the Ovsynch protocol (Brusveen et al., 2009).
However the increase in luteal regression did not result in increased conception rates in this instance (Brusveen et al., 2009). It has been reported that the binding of PGF2α to large luteal cells significantly increases from the early luteal phase to the late luteal phase
(Chegini et al., 1991). This suggests that a d 7 CL might not be as responsive to PGF2α as a fully mature CL. In the present study, more cows ovulated to hCG than GnRH which would then result in an increase in the proportion of cows with a d 7 CL at the time of
PGF2α administration. Another study investigating the efficacy of increasing the dosage of PGF2α from 500 to 750 µg of cloprostenol at the time of the second PGF2α of Double-
Ovsynch reported that luteal regression did not differ between dosage treatments
(Giordano et al., 2009). These studies thus suggest a possible advantage in administering two injections of PGF2α in order to increase luteal regression. Perhaps a second injection of PGF2α could have significantly reduced the proportion of cows treated with hCG failing to undergo luteal regression.
In the present study, the recommended single dose of dinoprost (25 mg) was used to induce luteal regression. However, dinoprost has a relatively short half-life of only a few minutes (EMEA, 1997b). It could be reasoned that because cows treated with hCG had a significantly greater number and volume of CL on d 7 of PO, that perhaps a luteolytic agent with a longer half-life such as cloprostenol (3 h; EMEA, 1997a) might have increased luteal regression in those cows due to a prolonged exposure of PGF2α to its receptors on the CL. However, a large study recently conducted in dairy cows reported that luteal regression was in fact greater in cows treated with dinoprost compared to cloprostenol regardless of number of CL at the time of treatment (Stevenson
59 Template Created By: Damen Peterson 2009 and Phatak, 2010). No differences in conception rates were detected between treatments
(Stevenson and Phatak, 2010). Cows with decreased body conditioning were also reported to have significantly increased luteal regression as compared to those with moderate conditioning (Stevenson and Phatak, 2010). In the present study, BCS did not affect luteal regression, but cows with decreased body conditioning and treated with
GnRH had significantly greater luteal regression than those with moderate body conditioning and treated with hCG.
Although the overall synchronization rate to PO was similar between treatments, it was significantly decreased in cows that had ovulated to hCG compared to GnRH. A recent study that replaced the first injection of GnRH of Ovsynch with hCG also reported a decreased synchronization rate in cows treated with hCG (Keskin et al., 2010). In this particular study, only ultrasonography was used to assess ovarian responses. Perhaps cows treated with hCG in this study were affected by increased concentrations of P4 at the time of the final GnRH as was the case in the current study. Research indicates that ovulation to the final GnRH of Ovsynch significantly decreases in cows with increased concentrations of P4 at that time (Stevenson, 2011). Since only a blood sample was taken in the present study at the onset of BO on d 17, it is not possible to give a true ovulation rate to the second injection of GnRH of PO, but it is likely that ovulation rates were decreased in cows originally treated with hCG. Days in milk also significantly affected the proportion of cows synchronized to PO, with hold-over cows having the greatest rate of synchrony. This could be explained by possibly more hold-over cows cycling at the onset of PO as compared to those in early lactation. Although cycling status was not determined before the onset of PO in the present study, it has been
60 Te mplate Created By: Da men Peterson 2009 r e p ort e d t h at 2 0 t o 3 0 % of d air y c o ws 6 0 t o 7 0 DI M ar e t y pi c all y a n o v ul ar ( G ü m e n e t al.,
2 0 0 3; L o p e z et al., 2 0 0 5). It w as r e p ort e d b y G ü m e n et al. ( 2 0 0 3) t h at a n o v ul ar c o ws h a d a si mil ar s y n c hr o ni z ati o n r at e t o o v ul ar c o ws w h e n s u bj e ct e d t o t h e O vs y n c h pr ot o c ol
( 9 1 %, n = 3 3 a n d 9 2 %, n = 1 1 7). I n c o ntr ast, St e v e ns o n ( 2 0 1 1) r e p ort e d a synchronization rate of only 55 % (n = 20) in anovular co ws subjected to the C O-Synch protocol. Perhaps synchronization rate in the present study was affected by more early l a ct ati o n c o ws b ei n g a n o v ul ar at t h e o ns et of P O as c o m p ar e d t o h ol d- o v er c o ws.
St u di es e v al u ati n g f olli c ul ar gr o wt h aft er a d mi nistr ati o n of h C G g e n er all y r e p ort d e cr e as e d ( Di a z et al., 1 9 9 8; K es ki n et al., 2 0 1 0) f olli cl e si z e, alt h o u g h n ot b y all ( D e
R e nsis et al., 1 9 9 9). I nt er esti n gl y, f olli c ul ar gr o wt h o nl y diff er e d o n d 7 of t h e pr es e nt study and by d 10 no difference was observed. In contrast, Keskin et al. (2010) replaced the first injection of Gn R H of Ovsynch with h C G and reported a tendency for a decrease i n t h e m a xi m al f olli cl e si z e at t h e ti m e of AI f or t h os e ori gi n all y tr e at e d wit h h C G.
F urt h er m or e, di a m et er of t h e l ar g est f olli cl e o n eit h er o v ar y i n t h e c urr e nt st u d y w as si g nifi c a ntl y aff e ct e d b y p arit y, wit h m ulti p ar o us c o ws c o nsist e ntl y h a vi n g gr e at er f olli cl e di a m et ers. T his p er h a ps c a n b e e x pl ai n e d b y t w o p ossi bl e s c e n ari os. T h e first is t h at pri mi p ar o us c o ws tr e at e d wit h h C G i n t his st u d y h a d t h e gr e at est c o n c e ntr ati o ns of
P4 on d 7 and 10 co mpared to any other subgroup (i.e., multiparous + h C G or Gn R H and pri miparous + Gn R H). Increased concentrations of P4 during follicular gro wth have b e e n r e p ort e d t o si g nifi c a ntl y d e cr e as e t h e si z e of t h e s u bs e q u e nt d o mi n a nt f olli cl e
( A d a ms et al., 1 9 9 2; Gi nt h er et al., 2 0 0 1). T his is c a us e d b y P 4 s u p pr essi n g cir c ul ati n g c o n c e ntr ati o ns of L H p ost-f olli c ul ar d e vi ati o n a n d r es ulti n g i n r e d u c e d gr o wt h of t h e d o mi n a nt f olli cl e ( Gi nt h er et al., 2 0 0 1). T h e s e c o n d p ossi bilit y f or t h e diff er e n c e i n
6 1 Template Created By: Damen Peterson 2009 follicle size could be attributed to the fact that multiparous cows generally have greater milk production and feed intake than primiparous cows (Nielsen et al., 2003). This could cause increased follicular size in multiparous cows due to increased metabolism of P4 through the increase in feed intake (Sangsritavong et al., 2002). Cows producing greater amounts of milk would have decreased circulating concentrations of P4 during follicular growth and result in an increase of LH pulses, causing increased growth of the dominant follicle (Wiltbank et al., 2006). However, milk production in the current study did not significantly affect follicle diameter. Follicle diameters in cows treated with hCG on d 0 of PO were then likely smaller due to both primiparous and multiparous cows treated with hCG having significantly greater concentrations of P4 than those treated with
GnRH. Likewise, similar follicle diameters detected on d 10 might be a result of only primiparous cows treated with hCG having significantly greater concentrations of P4.
Surprisingly, heifers treated with hCG in Experiment 2 had significantly greater follicle diameters on d 7 than did heifers treated with GnRH. In contrast, Dahlen et al.
(2008) measured the size of the largest follicle d 2 post-hCG and GnRH treatment in
Holstein heifers and reported significantly decreased follicle sizes for those treated with hCG. However by d 4, hCG-treated heifers only tended to have decreased follicle sizes when compared to those treated with GnRH. It is unclear why the heifers treated with hCG in the current study had a larger follicle diameter d 7 post-treatment. Because only
38.5% of heifers treated with GnRH had increased concentrations of P4 on d 7 (≥ 1 ng/mL), and follicle diameter on d 7 of PO was not affected by age or breed, perhaps more heifers treated with GnRH were in early metestrus on d 7 as compared to those treated with hCG. In addition, heifers treated with hCG and having decreased P4 (< 1
62 Template Created By: Damen Peterson 2009 ng/mL) on d7 had a significantly greater follicle diameter than heifers treated with GnRH and having decreased P4 on d 7, while no difference was indicated in follicle diameter between hCG and GnRH treated heifers having increased P4 on d 7. This suggests that there were possibly more heifers treated with hCG that were in proestrus on d 7 as compared to those treated with GnRH.
Concentrations of P4 in the current study were significantly decreased in hCG treated cows on d 17 (onset of BO) as compared to those treated with GnRH. However, when only compared between cows that were considered to be synchronized to PO, no difference was detected between treatments. This overall difference could be due to either failure of ovulation or possibly ovulation of a smaller dominant follicle after d 10.
A larger preovulatory follicle has been associated with increased concentrations of P4 on d 7 and 14 post-ovulation (Vasconcelos et al., 2001). Interestingly, no difference was observed between treatments in the diameter of the largest follicle on d 10. This could possibly be explained by looking specifically at primiparous cows treated with hCG.
These cows had the smallest follicle diameters as well as the greatest concentrations of
P4 on d 7 and 10 of PO. Perhaps decreased concentrations of P4 on d 17 in primiparous cows treated with hCG resulted in the ovulation of a smaller dominant follicle or possibly failure of ovulation due to increased concentrations of P4 on d 10. In comparison, concentrations of P4 on d 17 in Experiment 2 did not differ between treatments in heifers.
Conception rates in the current study did not differ between treatments for both cows and heifers. Likewise no difference was detected between treatments with the proportion of cows or heifers synchronized to PO. Previous literature has reported that as more cows are presynchronized before the onset of Ovsynch, conception rates are then
63 Template Created By: Damen Peterson 2009 significantly increased (Moreira et al., 2001; Navanukraw et al., 2004; Bello et al., 2006).
Unfortunately conception rates were significantly affected by an interaction of treatment by AI technician. Further investigation into a possible reason for this interaction has lead to inconclusive results. The significant interaction of treatment by technician then likely affected conception rates in cows considered to be synchronized to PO. For the heifers in
Experiment 2, conception rates were significantly affected by age of the heifer at the time of AI. A recent large scale study evaluating fertility in U.S. Holstein virgin heifers reported that age at AI significantly affects conception rates, with the optimum age of breeding being at 15 to 16 mo of age (Kuhn et al., 2006).
Conclusions
The results of the present study indicate that hCG had an increased capacity to induce ovulation in dairy cows as compared to those treated with GnRH. No difference was observed in the rates of luteal regression between treatments for both cows and heifers. However, a greater proportion of cows ovulating a follicle to hCG at the onset of
PO tended to fail to undergo luteal regression by d 3 post-PGF2α administration as compared to those ovulating a follicle to GnRH. The proportion of cows and heifers synchronized to PO did not differ between treatments, but cows ovulating to hCG had a significantly decreased synchronization rate to PO compared to those ovulating to GnRH.
In Experiment 2, many of the reproductive parameters measured in dairy heifers were affected by age and breed and was likely due to several heifers being pre-pubertal before the onset of PO. In conclusion, no advantage was identified in replacing the first
64 Template Created By: Damen Peterson 2009 injection of GnRH of PO with hCG for both cows and heifers on ovarian characteristics and responses, as well as subsequent fertility
65 Template Created By: Damen Peterson 2009
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Table 1 Plasma concentrations of progesterone and ovarian measurements taken in all cows during Pre-Ovsynch (d 0, 7, and 10)
Treatments Parameter GnRH hCG P-value No. of cows 91 92 hCG or GnRH injection (d 0) Progesterone, ng/mL 2.9 ± 0.4 3.5 ± 0.4 0.23 Increased progesterone1, (%) 57.1 63.0 0.38 Corpora lutea (CL), (n) 1.0 ± 0.1 1.2 ± 0.1 0.10 Total CL volume, cm3 3.6 ± 0.7 4.1 ± 0.7 0.60 Follicle diameter2, mm 13.4 ± 0.5 12.9 ± 0.5 0.49 PGF2α injection (d 7) Progesterone, ng/mL 3.0 ± 0.3 4.3 ± 0.3 < 0.01 Increased progesterone1, (%) 73.6 83.7 0.10 Corpora lutea (CL), (n) 1.3 ± 0.1 1.9 ± 0.1 < 0.001 Total CL volume, cm3 5.1 ± 0.6 7.5 ± 0.6 < 0.01 Follicle diameter2, mm 13.7 ± 0.4 12.7 ± 0.4 0.08 GnRH injection (d 10) Progesterone, ng/mL 0.5 ± 0.1 1.1 ± 0.1 < 0.01 Decreased progesterone3, (%) 81.3 69.6 0.07 Follicle diameter2, mm 14.9 ± 0.4 14.4 ± 0.4 0.32 1Percentage of cows with plasma progesterone ≥ 1 ng/mL. 2Diameter of largest follicle located on either ovary. 3Percentage of cows with plasma progesterone < 1 ng/mL on d 10.
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Table 2 Subset of cows that ovulated to injection of hCG or GnRH (d 0) and their plasma progesterone and ovarian measurements during Pre-Ovsynch (d 0, 7, and 10)
Treatment Parameter GnRH hCG P-value No. of cows 57 71 hCG or GnRH injection (d 0) Progesterone, ng/mL 1.6 ± 0.3 2.3 ± 0.3 0.13 Corpora lutea (CL), (n) 0.9 ± 0.1 1.2 ± 0.1 0.03 Total CL volume, cm3 3.0 ± 0.7 3.5 ± 0.6 0.60 Follicle diameter2, mm 14.1 ± 0.7 13.5 ± 0.6 0.51 PGF2α injection (d 7) Progesterone, ng/mL 3.8 ± 0.4 5.1 ± 0.3 0.01 Corpora lutea (CL), (n) 1.5 ± 0.1 2.0 ± 0.1 < 0.001 Total CL volume, cm3 6.3 ± 0.8 9.0 ± 0.7 0.01 Follicle diameter2, mm 13.8 ± 0.5 12.4 ± 0.5 0.03 GnRH injection (d 10) Progesterone, ng/mL 0.4 ± 0.2 1.2 ± 0.2 0.001 Decreased progesterone3, (%) 84.2 66.2 0.03 Follicle diameter2, mm 15.6 ± 0.5 14.8 ± 0.4 0.21 1Percentage of cows with plasma progesterone ≥ 1 ng/mL. 2Diameter of largest follicle located on either ovary. 3Percentage of cows with plasma progesterone < 1 ng/mL.
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Table 3 Ovulation, luteal regression, and synchronization rates in all cows and subset of cows that ovulated to hCG or GnRH injection during Pre-Ovsynch
All cows Subset of cows 1 Parameter GnRH hCG P-value GnRH hCG P-value No. of cows 91 92 56 71 Ovulation to hCG or GnRH2 (%) 62.2 77.2 0.03 - - 3 Luteal regression to PGF2α (%) 67.0 60.9 0.39 83.9 70.4 0.09 Early luteal regression4 (%) 19.8 15.2 0.45 - - Failed luteal regression5 (%) 12.1 23.9 0.04 16.1 29.6 0.09 Synchronization rate6 (%) 61.5 52.2 0.19 80.4 60.6 0.02 1Analysis of only cows that ovulated to injection of hCG or GnRH on d 0. 2Ovulation unable to be determined in one cow treated with GnRH on d 0. 3 Percentage of cows with plasma progesterone ≥ 1 ng/mL on d 7 and < 1 ng/mL on d 10. 4Percentage of cows with plasma progesterone ≥ 1 ng/mL on d 0 and < 1 ng/mL on d 7. 5Percentage of cows with plasma progesterone ≥ 1 ng/mL on d 7 and ≥ 1 ng/mL on d 10. 6Percentage of cows synchronized to Pre-Ovsynch. Defined as luteal regression from injection of PGF2α by d 10 and plasma progesterone ≥ 1 ng/mL on d 17.
Table 4 Conception rates and characteristics of progesterone of all cows measured during Breeding-Ovsynch
Treatments Parameter GnRH hCG P-value Conception rate1 (%) 32.2 25.0 0.92 (no./no.) (29/90) (23/92) Progesterone in all cows (d 17), ng/mL 3.5 ± 0.2 2.8 ± 0.2 0.02 (no.) (91) (92) Synchronized cows2, ng/mL 3.5 ± 0.2 3.2 ± 0.2 0.36 (no.) (56) (48) Increased progesterone3 (%) 86.8 82.6 0.43 (no./no.) (79/91) (76/92) 1Assessed at d 32 post-TAI (One cow culled in GnRH group before pregnancy diagnosis. 2Concentrations of progesterone on d 17 for cows synchronized to Pre-Ovsynch. 3Percentage of cows with plasma progesterone ≥ 1 ng/mL at time of first injection of GnRH of Breeding-Ovsynch (d 17).
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Table 5 Effect of parity on largest follicle diameter located on either ovary on d 0, 7, and 10, and concentrations of progesterone on d 17
Parity Parameter Primiparous Multiparous P-value Day 0 follicle diameter1, mm 12.0 ± 0.6 14.3 ± 0.5 0.01 (no.) (67) (116) Subset of cows 2, mm 14.3 ± 0.6 15.1 ± 0.5 0.35 (no.) (45) (82) Day 7 follicle diameter1, mm 12.6 ± 0.5 13.8 ± 0.4 0.05 (no.) (67) (116) Subset of cows 2, mm 12.3 ± 0.7 13.8 ± 0.5 0.04 (no.) (45) (82) Day 10 follicle diameter1, mm 14.0 ± 0.5 15.3 ± 0.4 0.04 (no.) (67) (116) Subset of cows 2, mm 14.4 ± 0.6 16.0 ± 0.4 0.03 (no.) (45) (82) Day 17 progesterone, ng/mL 2.9 ± 0.3 3.6 ± 0.2 0.08 (no.) (67) (116) Synchronized cows3, ng/mL 3.1 ± 0.2 3.6 ± 0.2 0.11 (no.) (37) (67) 1Diameter of largest follicle located on either ovary. 2Analysis of only cows that ovulated to injection of hCG or GnRH on d 0. 3Concentrations of progesterone for only cows synchronized to Pre-Ovsynch.
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Table 6 Plasma concentrations of progesterone and ovarian measurements taken in heifers during Pre-Ovsynch (d 0, 7, and 10)
Treatments Parameter GnRH hCG P-value No. of heifers1 26 25 hCG or GnRH injection (d 0) Progesterone, ng/mL 2.4 ± 0.6 3.3 ± 0.6 0.23 Increased progesterone2, (%) 57.7 56.0 0.87 PGF2α injection (d 7) Progesterone, ng/mL 2.9 ± 0.9 4.5 ± 0.9 0.14 Increased progesterone2, (%) 38.5 56.0 0.20 Low-Low3 (%) 26.9 20.0 0.52 No. of corpora lutea (CL), (no.) 0.9 ± 0.2 (21) 1.5 ± 0.2 (23) 0.03 Total CL volume, cm3 (no.) 2.5 ± 0.8 (21) 4.5 ± 0.8 (23) 0.08 Follicle diameter4, mm (no.) 10.9 ± 0.8 (21) 12.8 ± 0.7 (23) 0.05 GnRH injection (d 10) Progesterone, ng/mL 0.2 ± 0.1 0.4 ± 0.1 0.10 Decreased progesterone5, (%) 96.2 96.0 0.98 1Ovarian scans missing on 7 heifers (GnRH: n = 5; hCG: n = 2). 2Percentage of heifers with plasma progesterone ≥ 1 ng/mL. 3Percentage of heifers with plasma progesterone < 1 ng/mL on d 0 and 7. 4 Diameter of largest follicle located on either ovary. 5Percentage of heifers with plasma progesterone < 1 ng/mL on d 10.
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Table 7 Luteal regression, synchronization, and conception rates in heifers
Treatments Parameter GnRH hCG P-value No. of heifers 26 25 1 Luteal regression to PGF2α (%) 38.5 56.0 0.20 Early luteal regression2 (%) 30.8 24.0 0.59 Ovulated by d 10 (%) 47.6 47.8 0.99 (no./no.) (10/21) (11/23) Synchronization rate3 (%) 42.3 40.0 0.88 Progesterone d 17, ng/mL 4.7 ± 0.5 3.9 ± 0.5 0.32 Increased progesterone4 (%) 88.5 80.0 0.42 Conception rate5 (%) 30.8 36.0 0.65 1 Percentage of heifers with plasma progesterone ≥ 1 ng/mL on d 7 and < 1 ng/mL on d 10. 2Percentage of heifers with plasma progesterone ≥ 1 ng/mL on d 0 and < 1 ng/mL on d 7. 3Percentage of heifers synchronized to Pre-Ovsynch. Defined as luteal regression from injection of PGF2α by d 10 and plasma progesterone ≥ 1 ng/mL on d 17. 4Percentage of heifers with plasma progesterone ≥ 1 ng/mL at time of first injection of GnRH of Breeding-Ovsynch (d 17). 5Assessed at d 35 post-TAI.
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Table 8 Effect of age and breed of heifer on concentrations of progesterone, ovarian measurements, and synchronization and conception rates
Age of heifer at AI Breed of heifer Parameter < 15 mo ≥ 15 mo P- Holstein Jersey P- value value No. of heifers 26 25 41 10 Day 0 P41, ng/mL 2.0 ± 0.6 3.7 ± 0.6 0.03 2.5 ± 0.5 4.2 ± 0.9 0.13 Increased P42 (%) 38.5 76.0 < 0.01 51.2 80.0 0.03 Day 7 P4, ng/mL 2.4 ± 0.8 5.0 ± 1.0 0.02 2.5 ± 0.6 4.9 ± 1.3 0.09 Increased P42 (%) 38.5 56.0 0.11 41.5 70.0 0.07 Low-Low3 (%) 34.6 12.0 0.07 29.2 0.0 0.06 No. of CL4 1.1 ± 0.2 1.4 ± 0.2 0.20 1.0 ± 0.1 1.5 ± 0.2 0.07 (no. examined) (22) (22) (34) (9) CL volume, cm3 2.3 ± 0.8 4.8 ± 0.8 0.02 3.1 ± 0.6 5.0 ± 1.2 0.19 (no. examined) (22) (22) (34) (9) Follicle diameter 11.7 ± 11.5 ± 0.85 12.9 ± 10.8 ± 0.10 (mm) 0.8 1.0 0.6 1.1 (no. examined) (22) (22) (34) (9) Day 10 P4, ng/mL 0.3 ± 0.1 0.4 ± 0.1 0.45 0.3 ± 0.1 0.3 ± 0.2 0.99 Decreased P4 (%) 96.2 96.0 0.66 97.6 90.0 0.36 Luteal regression5 38.5 56.0 0.11 41.5 70.0 0.07 (%) Day 17 P4, ng/mL 3.5 ± 0.5 5.0 ± 0.5 0.05 4.0 ± 0.4 5.2 ± 0.8 0.19 Increased P42 (%) 69.2 100.0 0.01 82.9 90.0 0.55 Synchronized6 (%) 23.1 60.0 0.01 36.6 60.0 0.05 Conception rate7 (%) 19.2 48.0 0.04 36.6 20.0 0.53 1Progesterone. 2Percentage of heifers with progesterone ≥ 1 ng/mL. 3Percentage of heifers with plasma progesterone < 1 ng/mL on both d 0 and 7. 4Corpora lutea. 5Percentage of heifers with plasma progesterone ≥ 1 ng/mL on d 7 and < 1 ng/mL on d 10. 6Percentage of heifers synchronized to Pre-Ovsynch. 7Diagnosed at d 35 post-TAI.
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Pre-Ovsynch Breeding-Ovsynch
GnRH/hCG-7d-PGF2α-3d-GnRH-7d-GnRH -7d-PGF2α-48/56h-GnRH-16h-TAI
Treatment I (GnRH) 48 h (Heifers) Treatment II (hCG) 56 h (Cows)
BS+US* BS+US BS+US BS US=Ultrasound
D 0 D 7 D 10 D 17 BS=Blood Sample
Figure 1 Schematic of treatment protocols and sampling schedule for cows and heifers during Double-Ovsynch.
*Note only cows received ultrasound examination on d 0.
Figure 2 Concentrations of progesterone (P4) on d 10 of Pre-Ovsynch in dairy cows (LSMeans ± SEM; Experiment 1).
Numbers of cows are presented within each bar. a,bLSMeans within a category (all cows or cows that ovulated) with a different superscript differ (P < 0.05).
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Figure 3 Conception rates at d 32 post-TAI by treatment and AI technician in dairy cows (Experiment 1).
Numbers of cows are presented within each bar. a,bMeans within a category (cows treated with GnRH or hCG) with a different superscript differ (P < 0.05).
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Figure 4 Diameter of largest follicle located on either ovary on d 7 of Pre-Ovsynch in heifers (LSMeans ± SEM; Experiment 2).
Numbers of heifers are presented within each bar. a,bLSMeans within a category (< 1 or ≥ 1 ng/mL P4 on d7 of PO) with a different superscript differ (P < 0.05).
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