Bulgarian Journal of Veterinary Medicine, 2020, 23, No 1, 8088 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, 8088. 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 de- finitive method for examination of ovarian activity in water buffaloes. In some of the ovulating folli- cles the oocyte was absent during early puberty. The presence of corpora lutea confirmed the endo- crine 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 speci- mens 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.