Original Article HISTOLOGICAL CHARACTERISTICS of FOLLICULOGENESIS in MURRAH WATER BUFFALOES DURING the EARLY POSTPUBERTAL PERIOD

Bulgarian Journal of Veterinary Medicine, 2020, 23, No 1, 8088 ISSN 1311-1477; DOI: 10.15547/bjvm.2156

Original article

HISTOLOGICAL CHARACTERISTICS OF FOLLICULOGENESIS IN MURRAH WATER BUFFALOES DURING THE EARLY POSTPUBERTAL PERIOD

V. MANOV, V. PLANSKI & G. S. POPOV

Faculty of Veterinary Medicine, University of Forestry, Sofia, Bulgaria

Summary Manov, V., V. Planski & G. S. Popov, 2020. Histological characteristics of folliculogenesis in Murrah water buffaloes during the early postpubertal period. Bulg. J. Vet. Med., 23, No 1, 8088.

A characteristic feature of water buffalo heifers is that they approach breeding maturity later than bovine heifers. From a physiological and endocrinological view, this is related to a later puberty, which affects the overall reproductive performance of water buffalo. The aim of this study was to highlight some morphological characteristics of the water buffalo (Bubalus bubalis) ovaries in the early postpubertal period. The results showed active ovaries of the examined specimens. Some of the follicles had no oocyte, but were with normal structure and physiological activity. Histology is a definitive method for examination of ovarian activity in water buffaloes. In some of the ovulating follicles the oocyte was absent during early puberty. The presence of corpora lutea confirmed the endocrine maturity of the hypothalamus-pituitary-gonadal endocrine axis in 11–14 months old heifers despite the absence of oocytes. Key words: corpus luteum, estrus, follicle, ovary, ovulation, postpubertal period, water buffalo heifer

INTRODUCTION

Water buffalo heifers attain breeding ma- variable and is influenced by a wide vari- turity later than bovine heifers which is ety of factors, including climate, geo- attributed to later onset of puberty, affect- graphic area, breed, season of birth, and ing the overall reproductive performance. nutrition (Peeva et al., 1993). In dairy cattle, reproductive performance Research papers documenting the time is mainly affected by milk production of onset of puberty in water buffaloes vary (Slavkova et al., 2017), whereas live body considerably in their findings as to age weight gain and the season of puberty ranges at which this can be expected to onset are more important for reproductive occur. According to Jainudeen & Hafez management in water buffaloes (Planski et (1993), the river-type buffaloes approach al., 2017a). Water buffalo puberty age is puberty at 15–18 months of age, whereas V. Manov, V. Planski & G. S. Popov the swamp-type buffaloes exhibit normal 1.8±0.6 and 7.8±2.0 day respectively. The estrus at 21–24 months of age. The onset researchers noted that the dominant folli- of puberty in water buffalo is not currently cle diameter did not differ significantly clearly defined (Peeva et al., 1993; between the two models, but on the day of Borghese et al., 1996; Terzano et al., ovulation, dominant follicles were by 1997; Campanile et al., 2001; Singh et al., more than 4 mm greater than the subordi- 2010; Roy et al., 2016). Most of the find- nate follicles (Р≤0.05). ings are based on visual observations Follicular growth, recruitment, regres- rather than scientific methods that take sion and ovulation have been defined in into account the endocrine status of the 30 normally cycling Murrah water buffalo animals during this period. Using visual cows (Baruselli et al., 1997). One, two or observation, the earliest detected estrus in three waves of follicular dynamics were Bulgarian local water buffaloes has been found out. Structural development of the recorded at 21 months of age. Using ra- dominant follicle was longer in 2 and 3 dio-immunoassay (RIA) for hormone as- wave models without significant diffe- sessment in the same breed and crosses rence in the diameter. The number of fol- with Murrah, the first estrus has been en- licular waves and cycle duration depended docrinologically confirmed at 19.9 months on the activity of the corpus luteum. of age by Kanchev et al. (1989). In most Ovarian histology was used to define of the presented studies in the literature, the normal reproductive cycle and chan- onset of puberty has been calculated retro- ges associated with pregnancy in water spectively from the first calving date buffalo (Baithalua et al., 2013). The ova- (Borghese, 2005). According to Roy et al. ries and uteri of slaughterhouse specimens (2016) and Planski et al. (2017b), the use were examined. The classification of the of serum levels of progesterone to calcu- stage of cycle or pregnancy detected was late the onset of puberty is controversial. based on the maturity and development of Campanile et al. (2001) used both RIA the corpus luteum, superficial follicles and puberty confirmation and ultrasound foetal size. Large luteal cells were circular scanning to confirm the morphological in shape with central nuclei and multiple structure in the ovary responsible for the cytoplasmic lipid droplets. Small luteal high progesterone levels. cells were bundle shaped, with eccentric Taneja et al. (1996) characterised the irregular nuclei and a small number of growth and regression of ovarian follicles lipid droplets. The diameter of small in water buffalo, describing 7 full cycles luteal cells was approximately 12–23 µm, in 5 adult females using ultrasound scan- whereas the large cells were of signifi- ning once daily. Ovarian mapping was cantly larger size (25–55 µm). The ob- used, and a size of 14 mm for dominant served size increase was related to the follicles was specified. The results showed maturation of the corpus luteum (CL). The one or two follicular waves in each cycle, progesterone serum level was proportional each wave being characterised by the to the number of luteal cells present, presence of one dominant and multiple hence the high levels of serum progeste- subordinate follicles. In the one wave cy- rone recorded during diestrus and preg- cle, the cohort of follicles starts to de- nancy. The authors concluded that during velop at day 1.3±0.7 post oestrus. In the puberty, the mid diestrus CL was not fully two wave model, the cohorts start at mature and that, unlike cattle, was unable

BJVM, 23, No 1 81 Histological characteristics of folliculogenesis in Murrah water buffaloes during the early …. to supply sufficient progesterone to sup- were then sliced into 5 µm layers. The port pregnancy, supporting a hypothesis preparations were then denatured and of luteal insufficiency. stained using haematoxylin/eosin. The The aim of this study was to examine prepared slides were then examined inde- the ovaries of water buffalo heifers during pendently by two pathologists at the De- the early post pubertal period, using histo- partment of Internal Diseases, Pathology logical examination of the follicular dy- and Pharmacology at the Faculty of Vete- namics at different stages of the ovarian rinary Medicine, University of Forestry, cycle. Sofia, Bulgaria. The pathologists were unware of the history of the animals the samples were derived from. Specimens MATERIALS AND METHODS were examined by light microscopy with Animals and age of puberty onset different magnifications including immer- sion and a buildup camera. The software We had access to 76 Murrah water buffalo Euromex Bio Blue was used to prepare heifers assigned for slaughtering. Exami- and save the photographs. The total num- nation was carried out on animals born ber of examined slices was 48 (12 for during different seasons – once they were each examined animal). collected in the yard prior to slaughtering. The average age of the animals was 482 ±42 days. The examined 76 heifers were RESULTS observed visually for signs of estrus In all examined animals, the ovaries were 3 times a day. The observed signs were: active, with distinct cortical and medullary standing at mounting, presence of clear zones. In the periphery of the cortex, pri- mucous vaginal discharge and vulvar mordial follicles were noted between bun- swelling – according to the method de- dles of connective tissue. These follicles scribed by Suthar & Dhami (2010). contained an oocyte and one layer of Histological examination of the ovaries epithelial cells. The primary follicles are displayed on Fig. 1 and 2. They were out- Four heifers were randomly selected from lined with the presence of oocyte and one those recognised in estrus. They were at layer of epithelial cells around. Their av- the age of 11, 12, 14 and 15 months re- erage diameter, including the external spectively. To ascertain the exact age of layer was about 36 µm. the animals, the ear tag numbers of the In the deep layers of the parenchyma, heifers were checked against the Bulga- secondary follicles with developed oo- rian Food Safety Agency database. cytes were present and surrounded by a After slaughter, the ovaries of these thick protein membrane. Around this heifers were collected and preserved in membrane, cuboid and prismatic cells 3.7% formaldehyde. The histological ex- with a trophic function were present (co- amination of the collected specimens was rona radiata). Most externally was the carried out at the Institute of Experimental theca folliculi connective tissue mem- Morphology and Anthropology at the brane, which possessed epithelioid cells Bulgarian Academy of Sciences. The and small capillaries (Fig. 3). preparation was classical and the specimens were fixed in paraffin blocks, which

82 BJVM, 23, No 1 V. Manov, V. Planski & G. S. Popov

Fig. 1. Ovary from a water buffalo heifer – 11 months of age: cortical zone with growing follicles in different stages. Scale bar: 153.14 µm.

Fig. 2. Ovary from a water buffalo heifer – 12 months of age: primordial follicle (length 30.59 µm) and connective tissue cells. Scale bar: 17.30 µm. Tertiary follicles were present towards In the ovaries originating from the 15- the medulla of the ovarian parenchyma in month-old animal, atretic post-ovulation 14- and 15 month-old animals. These fol- follicles containing corpora lutea were licles had well developed follicular fluid present (Fig. 7 and 8). The average diame- cavities and were surrounded with follicu- ter of the tertiary follicle was about 420 lar epithelium. Surrounding the lumen, the µm, but most of them visualised on the oophoral cumulus was detected (Fig. 5 slides were ellipsoidal. Both longitudinal and 6). No visible oocytes were present in and transversal measurements were taken the tertiary follicles. to obtain the specified average diameter (Fig. 7).

BJVM, 23, No 1 83 Histological characteristics of folliculogenesis in Murrah water buffaloes during the early ….

The corpora lutea consisted of heifers. Histological findings demon- luteotrophs with central nuclei and cyto- strated different stages of follicular devel- plasmic lipid droplets. Small luteal cells opment and follicular maturation in the with bundle shape and eccentric nuclei 11-month-old heifer. Additionally, the were also visualised (Fig. 8). absence of active or regressing corpora lutea is suggestive of insufficient primary cycles occurring during the earlier stages DISCUSSION of puberty, and consequently in the post- The histological examination carried out pubertal period. provided significant information on the In many specimens, the observed folli- nature of folliculogenesis in water buffalo cles lacked a visible oocyte. This provides

Fig. 3. Ovary from a water buffalo heifer – 11 months of age: secondary follicle, theca granulosa and connective tissue external layer. Scale bar: 17.30 µm.мес

Fig. 4. Ovary from a water buffalo heifer – 14 months of age: ovarian cortex with tertiary follicle (length 36.67 µm), a few primary follicles in the periphery under the tunica albuginea. Scale bar: 66.67 µm.

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Fig. 5. Ovary from a water buffalo heifer – 14 months of age: tertiary follicle with oophoral cumulus. Scale bar: 66.67 µm.

Fig. 6. Ovary from a water buffalo heifer – 15 months of age: tertiary follicle with oophoral cumulus and absent oocyte. Scale bar: 66.67 µm. support to the hypothesis that insuffi- the size of the examined atretic follicles ciency of the cycle is present on an ova- was about 500 µm which is significantly rian level. This insufficiency appears to smaller compared to the size of the bovine last for a few cycles before and after pu- atretic follicles ≤5 mm as described by berty, or perhaps during the entire Rodgers & Irving-Rodgers (2010). parapubertal period. The rest of the cha- The presence of active corpora lutea racteristics of follicular morphology are on the histological sections of some of the consistent with those observed in sexually older heifers suggests that there was endo- mature animals. It must be stressed that crine induced control of luteal tissue re-

BJVM, 23, No 1 85 Histological characteristics of folliculogenesis in Murrah water buffaloes during the early ….

Fig. 7. Ovary from a water buffalo heifer – 15 months of age: atretic follicle, 500.47 µm (narrow arrow), tertiary follicle (thick arrow) and luteal tissue (star). Scale bar: 153.14 µm.

Fig. 8. Ovary from a water buffalo heifer – 15 months of age: corpus luteum. Luteotrophs with central nuclei and cytoplasmic lipid droplets (thick arrow), and small luteotrophs with bundle shaped nuclei (thin arrow). Scale bar: 66.67 µm. quired for successful cycling in these ani- The presented characteristics of the mals. Further study of the hormone pro- ovarian structures are comparable with files of water buffalo heifers would clarify available results (Taneja et al., 1996; the endocrine component of the cycle in Baithalua et al., 2013). The similarity is the postpubertal animals with follicles restricted to the type of the present struc- without oocytes. tures but not to the histological characte-

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ristics. Data similar to ours are presented ftp.fao.org/docrep/fao/010/ah847e/ah847e. in the work of Baithalua et al. (2013) pdf. which was focused on CL cell characteris- Borghese, A., M.T erzano, V. Barile, A. tics, but not on general histology and fol- Catalano, A. Maltati. 1996. Onset of pu- licular dynamics. berty in buffalo heifers in different feeding The presented structures in our results and management systems. In: Proceedings support the thesis for small tertiary follicle of the International Symposium on Buffalo Resources and Production Systems. Cairo, atresia previously stated by Fassi Fihri et Egypt, pp. 41–46. al. (2004) and Rodgers & Irving-Rodgers Campanile, G., R. Di Palo, B. Casparini, D. (2010). Occio & M. J. Zicarelli, 2001. Effects of Our findings are also similar with the early management system and subsequent average dimensions for the examined diet on growth and consumption in buffalo structures presented by Eurell & Frappier heifers. Livestock Production Science, 71, (2006) for the secondary follicle – 120 183–191. µm. The visual characteristics of the folli- Eurell, J. A. & B. Frappier, 2006. Textbook of cles also match the description of the cited Veterinary Histology, 6th edn, Blackwell authors. Publishing, USA. Fassi Fihri, A., K. H. Hajji & H. Lakhdissi, 2004. Histological exploration of follicular CONCLUSIONS population of the Moroccan bovine (Oul- mes-Zaers) breed. African Journal of Bio- Histology is the definitive method for technology, 3, 294–298. examination of ovarian activity in water buffalo. In some of the ovulating follicles Jainudeen, M. R. & E. S. E. Hafez, 1993. Re- productive cycles. In: Reproduction in the oocyte is absent during early puberty Farm Animals, 6th edn, Lea and Fabiger, in water buffalo heifers. The presence of Philadelphia, pp. 315–329. corpora lutea confirms the endocrine ma- Kanchev L., N. Bankov & L. Kostov, 1989. turity of the HPG endocrine axis in the Biology and Biotechnology of Reproduc- 11– 14 months old heifers despite the ab- tion, BAS Publishing, Sofia (BG). sence of oocytes. Peeva Ts., K. Vankov, M. Tsankova, O. Polihronov, A. Dragoev & A. Danev, REFERENCES 1993. All for Buffalo. Agrocomplex Ltd, Sofia (BG). Baithalua, R. K., S. K. Singha, Ch. Gupta, A. Planski, V., S. Yotov, M. Karadaev, Y. Ilieva, K. R., A. Saxenaa, Y. Kumara, R. Singh K. Hristov & D. Dimitrov, 2017a. Deter- & S. K. Agarwala. 2013. Cellular and mination of puberty onset in Bulgarian functional characterization of buffalo (Bu- Murrah buffalo heifers through blood pro- balus bubalis) corpus luteum during the gesterone analysis. International Journal estrous cycle and pregnancy. Animal Re- of Current Microbiology and Applied Sci- production Science, 140, 138–146. ences, 6, 308–314. Baruselli, P. S., R. G. Mucciolo, J. A. Visin- Planski, V., S. Yotov, P. Parvanov, M. Ka- tinz, W. G. Viana & R. P. Arruda, 1997. radaev, Y. Ilieva, K. Hristov & D. Dimit- Theriogenology, 47, 1531–1547. rov, 2017b. Influence of melatonin treat- Borghese, A. 2005. Buffalo Production and ment on puberty onset in buffalo heifers Research. Food and Agriculture Organi- from bulgarian murrah breed. Tradition zation. Rome, Italy, pp. 17–27, ftp:// and Modernity in Veterinary Medicine, 2, 56–60.

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