- Email: [email protected]
Seasonal variation in follicular dynamics of superovulated Indian water buffalo
SEASONAL VARIATION SUPEROWLATED M.
Taneja,Ia
IN FOLLICULAR INDIAN WATER S.M.
ToteyI
DYNAMICS BUFFALO
and
A. Ali2b
IEmbryo Biotechnology Laboratory Institute of Immunology, Aruna Asaf New Delhi 110 067 India 'Faculty of Natural Sciences, Jamia Millia New Delhi 110 025 India
National
Recieved
for
publication: Accepted:
OF
Febmaru
15, 1994
November
8,
Ali
Marg
Islamia
1994
ABSTRACT The ovaries of 5 buffalo were examined daily by ultrasound followed by beginning at Day 3 of the estrous cycle, superovulation between Days 11 and 13 of the cycle in both the Daily ultrasonographic observations wet cool and dry hot seasons. of the ovaries were recorded on a videotape and were used to assess the progression of both the large (dominant) and the next the numbers of to the large (sub-dominant) follicles as well as follicles in the small (4 to 6 mm), medium (7 to 10 mm) and large (> 10 mm) size categories in the 2 seasons before and during the of small (P < 0.05) superovulation treatment. Greater numbers and medium size follicles (P c 0.01) were available before the start of the superovulatory treatment in the buffalo during the dry hot season. The turnover of follicles from medium to large and was of higher size classes also occurred sooner (P < 0.01) magnitude (P < 0.05) during the treatment in the dry hot season. However, the number of corpora lutea at palpation per rectum (2.8 concentration (1.6 + + 0.7 vs 2.2 + 0.61, the serum progesterone and the yield of embryos on Day 6 (0.2 2 0.3 vs 1.4 + 0.1 ng/ml), 0.2 vs 0.6 i 0.2) did not differ significantly between the dry hot and the wet cool season. None of the embryos recovered during the dry hot season were transferable, which remains unexplained. Key
words:
buffalo, ultrasonography, ovary, superovulation, season
folliculogenesis,
Acknowledgements This work was supported by the Department of Biotechnology, and the Government of India. The authors thank Drs. 0. Singh with their help R. Pal, Immunoendocrinology Unit, NII, for technical assistance serum estimations. The progesterone also Ram Singh is provided by Inderjit, Radhey Shyam and ratefully acknowledged. 2 Correspondence and reprint requests. University of school of Pharmacy, bPresent address: The Madison WI 53706, USA. Wisconsin,
Theriogenology 43:451464, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 X/95/$1 0.00 SSDI 0093-691X(94)00036-V
452
Theriogenology INTRODUCTION
Buffalo are the mainstay of the dairy industry in both South and Southeast Asia. According to the FAO statistics for 1990 (II), India alone had 75 million buffalo out of a total world population of 140.86 million, providing more than 55% of the milk supply of the country. However, only 0.1% of the milk herd buffalo produce 3500 to 4000 kg of milk in a 305-d lactation period (23). The techniques of superovulation and embryo transfer coupled with progeny testing can play an important role in the propagation of these precious sources of germplasm. Superovulation aims at increasing the yield of viable embryos per flush and is a well-established technique in farm animals. In buffalo, various protocols for superovulation such as those used in cattle (PMSG or FSH administration beginning in the mid-luteal phase of the estrous cycle) have been tried over the past few years; however, the number of transferable embryos recovered has not been very encouraging (1,9,16,18,23,37). In India, buffalo are known to breed throughout the year; however, a distinct seasonal variation in the sexual rhythm under organized farm (3) and village conditions (29,36) has been larger number of buffalo exhibited reported. A significantly estrous symptoms and conceived during months with moderate temperatures, moderate humidity and shorter day lengths (36). To our knowledge, the effect of season on superovulation response in buffalo has not been studied to date. There are numerous factors which influence the superovulatory response and the yield of transferable embryos in cattle (2). One of the causes for animal variability in ovarian response to gonadotrophic stimulation in cattle is the stage of the estrous cycle when the superovulatory treatments are begun. It was found that beginning FSH treatment in the mid-luteal phase resulted in better response than beginning it in the early luteal phase (13,21), while there was no significant difference between treatments begun on each of Days 9 to 13 of the estrous cycle (6). The medium-size antral follicles, present in larger numbers in the mid-luteal phase, are known to respond favorably to exogenous gonadotrophin6 (25). Ultrasonographic monitoring of the ovaries has revealed that in cattle follicle growth occurs in 2 to 3 and sometimes 4 waves throughout the estrous cycle whereas precise information on the follicular (12,30,32), dynamics of buffalo either under field or experimental conditions is not available to date, other than for histologic observation of the ovaries (4) or palpation per rectum (34). Although the above studies provided important information, they did not sequentially monitor the population of follicles over a period of experiment was thus designed to study time. The present follicular growth before and during superovulation treatment by daily ultrasound monitoring of the ovaries of 5 buffalo during 2 fairly distinct seasons.
Theriogenology
453 MATERIALS AND METHODS
Animals
and Experimental
Design
Five nonlactating Murrah buffalo (Bubalus bubalis), 4 to 5 yr of age and having displayed at least I estrous cycle of normal duration (20 to 25 days), and weighing 450 to 550 kg, were used in the experiment. These buffalo were stall fed and were maintained at the National Institute of Immunology Research Farm Station (Faridabad, Haryana) . A once-daily bath was allowed to the buffalo throughout the year. The experiment was carried out on the same group of animals over 2 different seasons: the wet cool season of August to February (70 to 95% relative humidity, 36OC maximum atmospheric temperature, and 10.5 to 12.5 h of day length) and the dry hot season from April to June (35 to- 50% relative humidity, 45OC maximum atmospheric temperature, and 13.5 to 14.5 h of day length). For estrus synchronization the buffalo were treated twice with 500 ).lg, im cloprostenolC 11 d apart. Frequent daily observations were made to detect external signs of estrus, mainly mucous discharge and the response to placing of the hand on the rump and slight vulva1 massage (33). Starting on Day 3 of the estrous cycle (Day 0 = estrus), the ovaries of each buffalo were monitored daily for follicdular development by a real-time linear array ultrasound scanner equipped with a 5 MHz transrectal transducer. All observations were carried out by the same person (28) and were recorded on a videotape.e The tape was subsequently reviewed on the screen of an ultrasound scanner, and diagrams depicting the relative location of follicles 2 4 mm were made for each ovary and their growth and regression were Follicles were measured with a ruler individually monitored. calibrated against the in-built scale provided with the ultrasound unit (32). Although follicles of < 4 mm could be detected, their individual development could not be accurately followed and thus they were not included in the study. Superovulation treatment was begun between Days 11 and 13 of the estrous cycle with FSH-Pf (35 mg, im) administered in decreasing doses twice daily for 4 d (6, 5, 3.75, and 2.75 mg). For luteolysis the animals were treated with 500 pg, im at 48 h after the initiation of FSH treatment. A cloprostenolC breeding rest of at least 5 mo was provided to each buffalo between 2 consecutive superovulatory treatments. Four of the five buffalo were superovulated during the first wave of follicular The remaining buffalo was superovulated during development. second wave of follicular development in the wet cool season. The number of small (4 to 6 mm), medium (7 to 10 mm), and large ( > 10 mm) follicles were determined daily by ultrasonography until 1 d after the end of the superovulation treatment and recorded on a cEstrumate: Coopers Agropharm Inc., Ontario, Canada. dLS 300, Tokyo Keiki Co. Ltd., Tokyo 144, Japan. eVHS: National Panasonic, NV-E180SPX, Matsushita ndustrial Co. Ltd., Osaka, Japan. EFolltropin: Vetrepharm, London, Ontario, Canada.
Electric
Theriogenology videotape. The largest (FI) and second largest (F2) follicles were those that had the largest and the second largest diameters at the time of initiation of the superovulatory treatment (Figure 1) . Artificial insemination was performed at 48, 60 and 72 h after the cloprostenol treatment. Frozen-thawed Murrah buffalo bull semen was used for AI. Nonsurgical embryo recovery was performed on Day 6 using a two-way Foley catheter.9 Dulbecco' phosphate bufferred saline (DPBS) with 1.0% fetal calf serum ! (FCS) was used as a flushing medium. The number of corpora lutea (CL) were determined at palpation per rectum after embryo recovery on the same day. Progesterone
Assay
A peripheral blood sample was collected at the time of embryo recovery to determine serum progestrone concentrations for each buffalo. The serum was separated and stored at -20°c until assayed for progesterone. Progestrone estimations were made by using the WHO matched assay reagents. Then 25 pl of serum were vortexed with 2 ml freshly opened diethyl ether for 2 min at room temperature. The aqueous phase was frozen by cooling the tubes in liquid nitrogen vapors and the ether decanted in fresh tubes and allowed to evaporate at room temperature. Next 500 ~1 of steroid assay buffer (100 mM Phosphate buffer, 0.15 M NaCl, 0.01% Gelatin, pH 7.4) were added and the tubes heated to 40°C for 30 min. The tubes were then vigorously vortexed and an RIA was performed as described in the WHO Method Manual (27). Sensitivity of the assay was 12.2 pg/tube. All assays were performed in duplicate, and extraction controls were included to correct for interassay variations. The intra- and inter-assay coefficients of variation were 5 and 9%, respectively. The cross-reactivity of progesterone antisera was 1.6% with Id hydroxyprogesterone and 0.04% with 2Ocr di-hydroxyprogesterone. Validation
of Ultrasonographic
Observation
The validation of ultrasonographic measurements of follicular diameters was done with buffalo ovaries collected at slauqhter. Thirteen follicles 4 to 16 mm in size were dissected out and their diameters were measured with a vernier caliper after the trimminq of their stromal tissue. Each follicle was then placed in a water bath (22OC) and its antral cavity was measured with ultrasonography. There was a linear relationship between the diameters of follicles measured by ultrasonography and the diameters of the same follicles measured after dissection from the ovaries (y = -0.9 + 0.7x where y = follicle diameter by ultrasonography and x = follicle diameter after dissection; r = 0.9%; P < 0.001). On average, the diameters of dissected follicles were approximately 3 mm larger than the diameter of 9C.R. Bard,Inc., Murray Hill, NJ, USA. hGibco Laboratories, Grand Island, NY, USA.
455
Theriogenology
the antral cavity visible by ultrasonography, which has also been reported for cattle follicles (14,28). The thickness of the granulosa, theta interna, theta externa and a small amount of the stroma were the reasons reported for this size difference (32). Statistical
Analysis
The first day of superovulatory treatment was considered as Day 0. The data on follicular profiles were analyzed using twoway analysis of variance. The size of the dominant follicle (FI), sub-dominant follicle (F2), and the difference between the two (FI-F2) were compared between the wet cool season and the dry hot season. The follicles were classified according to size into small (4 to 6 mm), medium (7 to 10 mm) and large ( > 10 mm) categories and were compared separately between groups. The number of follicles within each class was also analyzed in a model, including the effect of day, with data sets before and during the superovulation being analyzed separately, while keeping Day 0 observations as the common element in both analyses. Appropriate interactions were also examined. The number of CL, number of total and transferable embryos, and number of unfertilized oocytes recovered as well as progesterone concentrations on Day 6 were compared between groups by analysis of variance. Data were transformed (logarithmic transformation) prior to statistical analysis (35). The Tadpole III1 statistical system was used for data analysis. RESULTS Superovulation
Response
Two buffalo (Nos. 1384 and 717) did not exhibit estrous symptoms after the superovulation treatment during the dry hot season; however, they were inseminated according to schedule and flushed thereafter on Day 6. Superovulatory estrus in the remaining 3 buffalo in the dry hot season appeared at an average of 33.3 + 0.8 h (Mean & SEM). In the wet cool season the superovulatory estrus appeared at an average of 40.4 + 8.1 h in 5 in the mean buffalo. There was also no significant difference number of CL evaluated at palpation per rectum (2.8 + 0.7 vs 2.2 + 0.6) or in the yield of the total number of embryos recovered on Day 6 (0.2 L 0.2 vs 0.6 + 0.2) between the wet cool season and the dry hot season. However, none of the embryos recovered in the dry hot season was transferable, including an unfertilized ova recovered from Buffalo 1384 and 2 degenerated embryos (I each) recovered from Buffalo 703 and 717. A single embryo recovered from Buffalo 6723 in the wet cool season was transferable (Figure 2). Mean Day 6 serum progesterone concentrations did not differ significantly between the wet cool and the dry Hot season (1.6 2 0.3 vs 1.4 + 0.1 ng/ml). lElsevier-BIOSOFT,
Cambridge, UK.
456
Figure
Theriogenology
1.
Ultrasonographic image of the left ovary of Buffalo 1384 before the start of superovulation treatment in the wet cool season. (Margins of the ovary are marked indicates the by small arrows, and the large arrow dominant follicle).
Theriogenology
457
WET
DRY
COOL
HOT
-
Days Fi .gure
before
and
after
start
of
SMALL
’ superovulatlon
2. Profiles of small (4-6 mm), medium (7-10 mm) and large ( z 10 mm) size follicles before and during the superovulation treatment in 5 buffalo in the wet cool season and the dry hot season. (Buffalo number; number of corpora lutea [CL] at palpation per rectum; and number of total [EM] and transferable embryos [TEI recovered on Day 6 are indicated in each panel; Day 0 = first day of superovulation treatment).
458
Theriogenology DRY
HOT
Fl F2 Fl
+
-4
-3
-2
-1
-
F2
0
of superovulation
Figure 3. Pattern of development of the largest (F1) and the second largest (F2) follicles and difference in their size (F1-F2) before the start of superovulation in 5 buffalo in the wet cool and the dry hot season. (Buffalo number and the day of the estrous cycle when superovulation was begun are indicated in each panel).
Theriogenology Follicular
459
Dynamics Before Superovulation
Treatment
The pattern of growth and regression of small (4 to 6 mm), medium (7 to 10 mm) and large ( > 10 mm) follicles before and during the superovulation treatment in individual buffalo superovulated in the wet cool season and the dry hot season are shown in Figure 2. Before the start of superovulation treatment, the means and the profiles of the mean number of small (P < 0.05) and medium size follicles (P < 0.01) differed, while the number of large size follicles did not differ between the 2 seasons. In the wet cool season the small follicles decreased in numbers from 13.2 + 2.4 to 8.4 + 2.1, while medium size follicles decreased from 2.4 + 0.4 to 1.8 + 0.6 before the start of treatment. In the dry hot season small follicles increased in numbers from 8.0 & 1.4 to 11.2 L 1.9 and medium size follicles increased from 3.0 5 0.7 to 5.2 f 1.6. The pattern of growth and regression of the largest and the second largest follicles and the difference between the size of the two, before the start of superovulation treatment, in individual buffalo superovulated in the wet cool season and the dry hot season are shown in Figure 3. There was no difference in the profile of the mean diameter of F1 follicles between the 2 seasons; however, the diameter increased sooner (at Day 3 before the start of treatment) in the dry hot season than in the wet cool season (day effect; P c 0.05). There was no difference in the profile of the mean diameter of F2 follicles between seasons; however, the diameter decreased sooner (at Day 2 before the start of treatment) in the dry hot season than in the wet cool season (day effect; P < 0.01). The mean difference in the diameters of F and F2 follicles increased progressively before treatment an ,: showed no difference between the seasons. Follicular
Dynamics During Superovulation
Treatment
The mean number of small follicles did not differ among the seasons; however, their number decreased progressively in the dry hot season, while it remained constant in the wet cool season P < 0.05) during the treatment and decreased (day effect; The mean number of medium size follicles did not thereafter. differ between the seasons; however, this size increased progressively until the end of treatment and decreased thereafter in the wet cool season as compared to the dry hot season, where the increase lasted until Day 3 of the treatment and declined thereafter (day effect; P < 0.05). The number of large follicles increased sooner (day effect; P c 0.01) in the dry hot season than in the wet cool season, and the magnitude of increase of the large follicles was higher (P c C.05) in the dry hot (1.6 + 0.2 to 5.2 + 0.9) than in the wet cool season (0.8 L 0.2 to 3.6 i. 0.7). Season-by-day interactions were neither significant in any of the 3 follicle size classes before and during treatment nor in the size of Fl and F2 follicles before the treatment. DISCUSSION The
time
of
onset
of
superovulatory
estrus
after
the
460
Theriogenology
prostaglandin administration was more variable in the wet cool season than in the dry hot season; however, 2 buffalo did not express estrus symptoms in the dry hot season, although they did ovulate and embryos could be recovered from them. In India and in other countries where the daylight increases with ambient temperature, ovarian activity and expression of estrus were both greater in buffalo in the cooler months. Higher serum prolactin levels were observed in buffalo during the summer months and was thought to be one of the reasons for the poor reproductive performance during these months, presumably due to the ovaries being refractory to the circulating gonadotrophins (17). The lowest sexual activity level was observed from March to July which was also the basis for the broad (3,5,10,29,36), classification of the 2 seasons in our present study. The number of CL as determined at palpation per rectum and by serum progesterone concentrations on Day 6 did not differ between the 2 seasons. It has been reported previously (8.38) that CL are fused or embedded deeply in the ovarian stroma in case of the buffalo and thus only in 25% of the buffalo did per rectum assessment coincided with the actual number of CL observed at slaughter (19). However, in the present study, the number of CL was not ascertained by ultrasonography and the animals were not slaughtered for this purpose. The yield of embryos was higher when the buffalo were superovulated in the dry hot season than in the wet cool season; however, the dry hot season appears to be detrimental to the quality of embryos. This is not likely to be due to the effect of repeated gonadotrophin treatments, since a sufficient rest from breeding was provided to the animals between 2 consecutive It has been reported previously superovulation treatments. (22,24) that with repeated superovulation of high responding buffalo, the responses can be further improved with better hormone preparations. Before the start of superovulation treatment, the recruitment of small and medium size follicles differed between the wet cool and dry hot seasons, with greater numbers of follicles in these classes becoming atretic in the wet cool season. It aonears from the profiles of srowth and resression of the largest *follicle (FI) before supero;ulation treatment that the functional status of the dominant follicle in buffaloes does not differ between the seasons, although the size of the largest follicle alone apparently is not a satisfactory criterion for assessing follicular dominance (I4), as it has been demonstrated that morphologically dominant follicles have different levels of atresia during the growing and regressing phases of development (15). However, such studies have required careful dissection of the ultrasonographically detected dominant follicle in various stages of development for histological and biochemical analysis. The diameter of the second largest (F2) follicle decreased constantly in both seasons. The increase in diameter of the F and decrease in diameter of the F2 follicle occurre a follicle
461
Theriogenology
sooner in the dry hot season than in the wet cool season; however, there was a parallel increase in the difference between the diameters of these (F1 and F2) follicles before the start of superovulation in both the seasons, thereby suggesting the presence of functional dominance in this group of buffalo that large irrespective of season. It has been demonstrated follicles present on the ovaries on Day 7 or Day 9 of the estrous cycle in cattle heifers are functional, since they ovulate following prostaglandin-induced luteolysis (7,20 31). In the present study, 4 of the 5 buffalo were superovulated during the first wave of follicular development. One buffalo was superovulated in the second wave during the wet cool season. However, functional dominance and its effect on superovulation still need to be evaluated in the buffalo. Compared with that of the wet cool season, the dry hot season was marked with a progressive decrease in the number of small follicles, a rapid disappearance of follicles from the medium size class, and a rapid increase in large size follicles (Day 3 of treatment) during the superovulation treatment. This suggests that the process of recruitment and passage of follicles from smaller to larger size classes is better in the dry hot explaining the season than in the wet cool season, possibly better yield of embryos in the dry hot season. However, these embryos were not of transferable quality, and the reason for this could not be ascertained. This study needs to be conducted on a larger group of animals; however, it is difficult to find cyclic during the dry hot buffalo throughout the year, especially months. In cattle more than 80% of the ovulations are reported to occur over a 2-d period in response to superovulation, and it has been suggested that the remaining ovulations represent follicles which could be less mature and which did not respond normally to the LH surge (26,39). In superovulated buffalo the timing of ovulations in response to the LH surge and its effect on oocyte maturation with regard to recovery of good quality embryos is yet to be studied. REFERENCES 1.
2. 3. 4.
Ambrose JD, Singla SK, Jailkhani S, Prakash BS, Madan ML. Superovulation response in buffaloes (Bubalus bubalis) to Theriogenology different regimes of Folltropin. treatment 1991;35:181 abstr. in superovulation of cattle. Armstrong DT. Recent advances Theriogenology 1993;39:7-24. buffaloes. Basu S. Seasonal variation of fertility in murrah Indian Vet J 1962;39:433-438. Dane11 B. Oestrous Behaviour,Ovarian Morphology and Cyclical Pattern in Variation in Follicular System and Endocrine Water Buffalo Heifers (Ph.D. dissertation). Uppsala, Sveriges Lantbruksuniversitet,l987;54-94.
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Theriogenology Dobson H, Kamonpatana M. A review of female cattle reproduction with special reference to comparison between buffaloes, cows and zebu. J Reprod Fzrtil 1986;77: l-36. Donaldson LE. The day of the estrous cycle that FSH is started and superovulation in cattle. Theriogenology 1984; 22:97-99. Driancourt MA, Thatcher WW, Terqui M, Andrieu D. Dynamics of ovarian follicular development in cattle during the estrous cycle, early pregnancy and in response to PMSG. Dom Anim Endocrinol 1991;8:209-221. Drost M, Cripe WS. Superovulation and embryo transfer in buffaloes (Bubalus bubalis). Proc First World Buffalo Congr, 1985;3:498-505. Drost M, Elsden PR. Blastocyst development in the water buffalo (Bubalus bubalis).Theriogenology 1985;23:191 abstr. El-Foulv MA. Kotbv EA. El-Sobhv AE. The functional reproductive peak in Egyptian buffalo cows is related to day length and ambient temperature. Arch Vet Ital 1976; 7:123-129. FAO Buffalo Statistics. In: Buffalo Production Around the World-Problems and Prospects. Bangkok, International Buffalo Information Center, Kasetsart University,l991;6-7. Ginther OJ, Kastelic JP, Knopf L. Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Therioqenoloqv 1989;32:787-795. Williams DH; Duff; 0, Goulding D, Boland MP, Roche JF. Superovulation in heifers qiven FSH initiated either at day 2 -or day 10 of the estrous cycle. Theriogenology 1990; 34~767-770. Guibault LA, Grass0 F, Lussier JG, Rouillier P, Matton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 1991;91:8i-89. Guilbault LA, Rouillier P, Matton P, Glencross RG, Beard AJ, Knight PG. Relationships between the level of atresia and inhibin contents (crsubunit and cc-is dimer) in morphologically dominant follicles during their growing and regressing phases of development in cattle. Biol Reprod 1993;48: 268-276. Jain GC, Kumar P, Khanna S, Elsden PR, Lohan IS. Calves born from embryo transfer technology in buffaloes. Intl J Anim Sci 1990;5:203-208. Janakiraman K. Control and optimising of reproduction cycle in buffaloes. Proc Buffalo Reprod Arti Insem Seminar, FAO/ SIDA/Govt of India, Karnal, 1979;220-225. Karaivanov C. Comparative studies on the superovulatory effect of PMSG and FSH in water buffalo (Bubalus bubalis) . Theriogenology 1986;26:51-59. Karaivanov C, Kachena D, Petrov M, Vlahov K, Sapundjiev E. Superovulatory response of river buffalo (Bubalus bubalis) . Theriogenology 1990;33:453-464.
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20. Kastelic JP, Knopf L, Ginther OJ. Effect of day of prostaglandin F &;?yP";; o~,s~~e,c,'i~i~dRe~~~~lo~~~n~99~~ the ovulatory 23:169-180. 21. Lindsell CE, Murphy RD, Mapletoft RJ. Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle. Theriogenology 1986; 26:209-219. buffalo: 22. Misra AK. Superovulation and embryo transfer in Progress, problems and future prospects in India. Buffalo J 1993;1:13-24. Kasiraj R, 23. Misra AK, Joshi BV, Agrawala PL, Sivaiah S, RangareddiNS, Siddiqui MU. Multiple ovulation and embryo transfer in Indian buffalo (Bubalus bubalis). Theriogenology 1990;33:1131-1141. 24. Misra AK, Joshi BV, Kasiraj R, Sivaiah S, Ranqareddi NS. superovulatory regimen buffaloImproved for (Bubalus bubalis) . Theriogenology 1991;35:245 abstr. of 25. Moor RM, Kruip AM Th, Green D. Intraovarian control folliculogenesis: Limits to superovulation? Theriogenology 1984;21:103-116. populations during the 26. Pierson RA, Ginther OJ. Follicular estrous cycle in heifers. Anim Reprod Sci 1988;16:81-95. of 27. Progesterone assay. In: WHO Programme for the Provision Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology Method Manual. Geneva, WHO, 1989; 67-79. of 28. Quirk SM, Hickey GJ, Fortune JE. Growth and regression ovarian follicles during the follicular phase of the oestrus cycle in heifers-undergoing spontaneous and PGF2,induced luteolysis. J Reprod Fertil 1986;77:211-219. village herds of Indian water 29. Rao AVN, Rao CS. Oestrus-in buffaloes. Indian Vet J 1970;47:742-748. Keenan L, 30. Savio JD, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J Reprod Fertil 1988;83:663-671. MR, Roche JF. 31. Savio JD, Boland MP, Hynes N, Mattiacci heifers Will the first dominant follicle of the cycle of ovulate following luteolysis on day 7? Theriogenology 1990; 33~677-688. the 32. Sirois J, Fortune JE. Ovarian follicular dynamics during monitored real-time cycle in heifers estrous by ultrasonography. Biol Reprod 1988;39:308-317. 33. Sinsh G, Sinsh GB, Sharma SS, Sharma RD. Studies on oestrous symptoms of buffalo heifers. Theriogenology 1984;21:849-858. follicular on 34. Sinqh G, Singh GB, Sharma ss. Studies patterns in buffalo-heifers. Theriogenology 1984;22:453-462. The Iowa Methods, 35. Snedecor GW, Cochran WG. Statistical State University Press,1967;329-330. and SP, Jain LS, Gupta HK, Bhatia JS. Oestrus 36. Tailor conditions. under village conception rates in buffaloes Indian J Anim Sci 1990;60:1020-1021.
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37. Taneja M. Totev SM. Pawshe CH, Ali A, Sinsh G. Effect of GnRH- incorporation into superovulation regimen for buffalo (Bubalus bubalis) . Proc 12th Intl Congr Anim Reprod, Hague, 1992;1:278-280. Wright Jr RW, Mehmood A, Baig SM. Endocrine 38 Ullah N, profiles in relation to ovarian response, recovery rate and quality of embryos in Nili-Ravi buffaloes treated with FSH. Buffalo J 1992;1:47-56. and onset 39 Yadav MC, Walton JS, Leslie KE. Timing of the duration of ovulation in superovulated beef heifers. Theriogenology 1986;26:509-521.
Taneja,Ia
IN FOLLICULAR INDIAN WATER S.M.
ToteyI
DYNAMICS BUFFALO
and
A. Ali2b
IEmbryo Biotechnology Laboratory Institute of Immunology, Aruna Asaf New Delhi 110 067 India 'Faculty of Natural Sciences, Jamia Millia New Delhi 110 025 India
National
Recieved
for
publication: Accepted:
OF
Febmaru
15, 1994
November
8,
Ali
Marg
Islamia
1994
ABSTRACT The ovaries of 5 buffalo were examined daily by ultrasound followed by beginning at Day 3 of the estrous cycle, superovulation between Days 11 and 13 of the cycle in both the Daily ultrasonographic observations wet cool and dry hot seasons. of the ovaries were recorded on a videotape and were used to assess the progression of both the large (dominant) and the next the numbers of to the large (sub-dominant) follicles as well as follicles in the small (4 to 6 mm), medium (7 to 10 mm) and large (> 10 mm) size categories in the 2 seasons before and during the of small (P < 0.05) superovulation treatment. Greater numbers and medium size follicles (P c 0.01) were available before the start of the superovulatory treatment in the buffalo during the dry hot season. The turnover of follicles from medium to large and was of higher size classes also occurred sooner (P < 0.01) magnitude (P < 0.05) during the treatment in the dry hot season. However, the number of corpora lutea at palpation per rectum (2.8 concentration (1.6 + + 0.7 vs 2.2 + 0.61, the serum progesterone and the yield of embryos on Day 6 (0.2 2 0.3 vs 1.4 + 0.1 ng/ml), 0.2 vs 0.6 i 0.2) did not differ significantly between the dry hot and the wet cool season. None of the embryos recovered during the dry hot season were transferable, which remains unexplained. Key
words:
buffalo, ultrasonography, ovary, superovulation, season
folliculogenesis,
Acknowledgements This work was supported by the Department of Biotechnology, and the Government of India. The authors thank Drs. 0. Singh with their help R. Pal, Immunoendocrinology Unit, NII, for technical assistance serum estimations. The progesterone also Ram Singh is provided by Inderjit, Radhey Shyam and ratefully acknowledged. 2 Correspondence and reprint requests. University of school of Pharmacy, bPresent address: The Madison WI 53706, USA. Wisconsin,
Theriogenology 43:451464, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 X/95/$1 0.00 SSDI 0093-691X(94)00036-V
452
Theriogenology INTRODUCTION
Buffalo are the mainstay of the dairy industry in both South and Southeast Asia. According to the FAO statistics for 1990 (II), India alone had 75 million buffalo out of a total world population of 140.86 million, providing more than 55% of the milk supply of the country. However, only 0.1% of the milk herd buffalo produce 3500 to 4000 kg of milk in a 305-d lactation period (23). The techniques of superovulation and embryo transfer coupled with progeny testing can play an important role in the propagation of these precious sources of germplasm. Superovulation aims at increasing the yield of viable embryos per flush and is a well-established technique in farm animals. In buffalo, various protocols for superovulation such as those used in cattle (PMSG or FSH administration beginning in the mid-luteal phase of the estrous cycle) have been tried over the past few years; however, the number of transferable embryos recovered has not been very encouraging (1,9,16,18,23,37). In India, buffalo are known to breed throughout the year; however, a distinct seasonal variation in the sexual rhythm under organized farm (3) and village conditions (29,36) has been larger number of buffalo exhibited reported. A significantly estrous symptoms and conceived during months with moderate temperatures, moderate humidity and shorter day lengths (36). To our knowledge, the effect of season on superovulation response in buffalo has not been studied to date. There are numerous factors which influence the superovulatory response and the yield of transferable embryos in cattle (2). One of the causes for animal variability in ovarian response to gonadotrophic stimulation in cattle is the stage of the estrous cycle when the superovulatory treatments are begun. It was found that beginning FSH treatment in the mid-luteal phase resulted in better response than beginning it in the early luteal phase (13,21), while there was no significant difference between treatments begun on each of Days 9 to 13 of the estrous cycle (6). The medium-size antral follicles, present in larger numbers in the mid-luteal phase, are known to respond favorably to exogenous gonadotrophin6 (25). Ultrasonographic monitoring of the ovaries has revealed that in cattle follicle growth occurs in 2 to 3 and sometimes 4 waves throughout the estrous cycle whereas precise information on the follicular (12,30,32), dynamics of buffalo either under field or experimental conditions is not available to date, other than for histologic observation of the ovaries (4) or palpation per rectum (34). Although the above studies provided important information, they did not sequentially monitor the population of follicles over a period of experiment was thus designed to study time. The present follicular growth before and during superovulation treatment by daily ultrasound monitoring of the ovaries of 5 buffalo during 2 fairly distinct seasons.
Theriogenology
453 MATERIALS AND METHODS
Animals
and Experimental
Design
Five nonlactating Murrah buffalo (Bubalus bubalis), 4 to 5 yr of age and having displayed at least I estrous cycle of normal duration (20 to 25 days), and weighing 450 to 550 kg, were used in the experiment. These buffalo were stall fed and were maintained at the National Institute of Immunology Research Farm Station (Faridabad, Haryana) . A once-daily bath was allowed to the buffalo throughout the year. The experiment was carried out on the same group of animals over 2 different seasons: the wet cool season of August to February (70 to 95% relative humidity, 36OC maximum atmospheric temperature, and 10.5 to 12.5 h of day length) and the dry hot season from April to June (35 to- 50% relative humidity, 45OC maximum atmospheric temperature, and 13.5 to 14.5 h of day length). For estrus synchronization the buffalo were treated twice with 500 ).lg, im cloprostenolC 11 d apart. Frequent daily observations were made to detect external signs of estrus, mainly mucous discharge and the response to placing of the hand on the rump and slight vulva1 massage (33). Starting on Day 3 of the estrous cycle (Day 0 = estrus), the ovaries of each buffalo were monitored daily for follicdular development by a real-time linear array ultrasound scanner equipped with a 5 MHz transrectal transducer. All observations were carried out by the same person (28) and were recorded on a videotape.e The tape was subsequently reviewed on the screen of an ultrasound scanner, and diagrams depicting the relative location of follicles 2 4 mm were made for each ovary and their growth and regression were Follicles were measured with a ruler individually monitored. calibrated against the in-built scale provided with the ultrasound unit (32). Although follicles of < 4 mm could be detected, their individual development could not be accurately followed and thus they were not included in the study. Superovulation treatment was begun between Days 11 and 13 of the estrous cycle with FSH-Pf (35 mg, im) administered in decreasing doses twice daily for 4 d (6, 5, 3.75, and 2.75 mg). For luteolysis the animals were treated with 500 pg, im at 48 h after the initiation of FSH treatment. A cloprostenolC breeding rest of at least 5 mo was provided to each buffalo between 2 consecutive superovulatory treatments. Four of the five buffalo were superovulated during the first wave of follicular The remaining buffalo was superovulated during development. second wave of follicular development in the wet cool season. The number of small (4 to 6 mm), medium (7 to 10 mm), and large ( > 10 mm) follicles were determined daily by ultrasonography until 1 d after the end of the superovulation treatment and recorded on a cEstrumate: Coopers Agropharm Inc., Ontario, Canada. dLS 300, Tokyo Keiki Co. Ltd., Tokyo 144, Japan. eVHS: National Panasonic, NV-E180SPX, Matsushita ndustrial Co. Ltd., Osaka, Japan. EFolltropin: Vetrepharm, London, Ontario, Canada.
Electric
Theriogenology videotape. The largest (FI) and second largest (F2) follicles were those that had the largest and the second largest diameters at the time of initiation of the superovulatory treatment (Figure 1) . Artificial insemination was performed at 48, 60 and 72 h after the cloprostenol treatment. Frozen-thawed Murrah buffalo bull semen was used for AI. Nonsurgical embryo recovery was performed on Day 6 using a two-way Foley catheter.9 Dulbecco' phosphate bufferred saline (DPBS) with 1.0% fetal calf serum ! (FCS) was used as a flushing medium. The number of corpora lutea (CL) were determined at palpation per rectum after embryo recovery on the same day. Progesterone
Assay
A peripheral blood sample was collected at the time of embryo recovery to determine serum progestrone concentrations for each buffalo. The serum was separated and stored at -20°c until assayed for progesterone. Progestrone estimations were made by using the WHO matched assay reagents. Then 25 pl of serum were vortexed with 2 ml freshly opened diethyl ether for 2 min at room temperature. The aqueous phase was frozen by cooling the tubes in liquid nitrogen vapors and the ether decanted in fresh tubes and allowed to evaporate at room temperature. Next 500 ~1 of steroid assay buffer (100 mM Phosphate buffer, 0.15 M NaCl, 0.01% Gelatin, pH 7.4) were added and the tubes heated to 40°C for 30 min. The tubes were then vigorously vortexed and an RIA was performed as described in the WHO Method Manual (27). Sensitivity of the assay was 12.2 pg/tube. All assays were performed in duplicate, and extraction controls were included to correct for interassay variations. The intra- and inter-assay coefficients of variation were 5 and 9%, respectively. The cross-reactivity of progesterone antisera was 1.6% with Id hydroxyprogesterone and 0.04% with 2Ocr di-hydroxyprogesterone. Validation
of Ultrasonographic
Observation
The validation of ultrasonographic measurements of follicular diameters was done with buffalo ovaries collected at slauqhter. Thirteen follicles 4 to 16 mm in size were dissected out and their diameters were measured with a vernier caliper after the trimminq of their stromal tissue. Each follicle was then placed in a water bath (22OC) and its antral cavity was measured with ultrasonography. There was a linear relationship between the diameters of follicles measured by ultrasonography and the diameters of the same follicles measured after dissection from the ovaries (y = -0.9 + 0.7x where y = follicle diameter by ultrasonography and x = follicle diameter after dissection; r = 0.9%; P < 0.001). On average, the diameters of dissected follicles were approximately 3 mm larger than the diameter of 9C.R. Bard,Inc., Murray Hill, NJ, USA. hGibco Laboratories, Grand Island, NY, USA.
455
Theriogenology
the antral cavity visible by ultrasonography, which has also been reported for cattle follicles (14,28). The thickness of the granulosa, theta interna, theta externa and a small amount of the stroma were the reasons reported for this size difference (32). Statistical
Analysis
The first day of superovulatory treatment was considered as Day 0. The data on follicular profiles were analyzed using twoway analysis of variance. The size of the dominant follicle (FI), sub-dominant follicle (F2), and the difference between the two (FI-F2) were compared between the wet cool season and the dry hot season. The follicles were classified according to size into small (4 to 6 mm), medium (7 to 10 mm) and large ( > 10 mm) categories and were compared separately between groups. The number of follicles within each class was also analyzed in a model, including the effect of day, with data sets before and during the superovulation being analyzed separately, while keeping Day 0 observations as the common element in both analyses. Appropriate interactions were also examined. The number of CL, number of total and transferable embryos, and number of unfertilized oocytes recovered as well as progesterone concentrations on Day 6 were compared between groups by analysis of variance. Data were transformed (logarithmic transformation) prior to statistical analysis (35). The Tadpole III1 statistical system was used for data analysis. RESULTS Superovulation
Response
Two buffalo (Nos. 1384 and 717) did not exhibit estrous symptoms after the superovulation treatment during the dry hot season; however, they were inseminated according to schedule and flushed thereafter on Day 6. Superovulatory estrus in the remaining 3 buffalo in the dry hot season appeared at an average of 33.3 + 0.8 h (Mean & SEM). In the wet cool season the superovulatory estrus appeared at an average of 40.4 + 8.1 h in 5 in the mean buffalo. There was also no significant difference number of CL evaluated at palpation per rectum (2.8 + 0.7 vs 2.2 + 0.6) or in the yield of the total number of embryos recovered on Day 6 (0.2 L 0.2 vs 0.6 + 0.2) between the wet cool season and the dry hot season. However, none of the embryos recovered in the dry hot season was transferable, including an unfertilized ova recovered from Buffalo 1384 and 2 degenerated embryos (I each) recovered from Buffalo 703 and 717. A single embryo recovered from Buffalo 6723 in the wet cool season was transferable (Figure 2). Mean Day 6 serum progesterone concentrations did not differ significantly between the wet cool and the dry Hot season (1.6 2 0.3 vs 1.4 + 0.1 ng/ml). lElsevier-BIOSOFT,
Cambridge, UK.
456
Figure
Theriogenology
1.
Ultrasonographic image of the left ovary of Buffalo 1384 before the start of superovulation treatment in the wet cool season. (Margins of the ovary are marked indicates the by small arrows, and the large arrow dominant follicle).
Theriogenology
457
WET
DRY
COOL
HOT
-
Days Fi .gure
before
and
after
start
of
SMALL
’ superovulatlon
2. Profiles of small (4-6 mm), medium (7-10 mm) and large ( z 10 mm) size follicles before and during the superovulation treatment in 5 buffalo in the wet cool season and the dry hot season. (Buffalo number; number of corpora lutea [CL] at palpation per rectum; and number of total [EM] and transferable embryos [TEI recovered on Day 6 are indicated in each panel; Day 0 = first day of superovulation treatment).
458
Theriogenology DRY
HOT
Fl F2 Fl
+
-4
-3
-2
-1
-
F2
0
of superovulation
Figure 3. Pattern of development of the largest (F1) and the second largest (F2) follicles and difference in their size (F1-F2) before the start of superovulation in 5 buffalo in the wet cool and the dry hot season. (Buffalo number and the day of the estrous cycle when superovulation was begun are indicated in each panel).
Theriogenology Follicular
459
Dynamics Before Superovulation
Treatment
The pattern of growth and regression of small (4 to 6 mm), medium (7 to 10 mm) and large ( > 10 mm) follicles before and during the superovulation treatment in individual buffalo superovulated in the wet cool season and the dry hot season are shown in Figure 2. Before the start of superovulation treatment, the means and the profiles of the mean number of small (P < 0.05) and medium size follicles (P < 0.01) differed, while the number of large size follicles did not differ between the 2 seasons. In the wet cool season the small follicles decreased in numbers from 13.2 + 2.4 to 8.4 + 2.1, while medium size follicles decreased from 2.4 + 0.4 to 1.8 + 0.6 before the start of treatment. In the dry hot season small follicles increased in numbers from 8.0 & 1.4 to 11.2 L 1.9 and medium size follicles increased from 3.0 5 0.7 to 5.2 f 1.6. The pattern of growth and regression of the largest and the second largest follicles and the difference between the size of the two, before the start of superovulation treatment, in individual buffalo superovulated in the wet cool season and the dry hot season are shown in Figure 3. There was no difference in the profile of the mean diameter of F1 follicles between the 2 seasons; however, the diameter increased sooner (at Day 3 before the start of treatment) in the dry hot season than in the wet cool season (day effect; P c 0.05). There was no difference in the profile of the mean diameter of F2 follicles between seasons; however, the diameter decreased sooner (at Day 2 before the start of treatment) in the dry hot season than in the wet cool season (day effect; P < 0.01). The mean difference in the diameters of F and F2 follicles increased progressively before treatment an ,: showed no difference between the seasons. Follicular
Dynamics During Superovulation
Treatment
The mean number of small follicles did not differ among the seasons; however, their number decreased progressively in the dry hot season, while it remained constant in the wet cool season P < 0.05) during the treatment and decreased (day effect; The mean number of medium size follicles did not thereafter. differ between the seasons; however, this size increased progressively until the end of treatment and decreased thereafter in the wet cool season as compared to the dry hot season, where the increase lasted until Day 3 of the treatment and declined thereafter (day effect; P < 0.05). The number of large follicles increased sooner (day effect; P c 0.01) in the dry hot season than in the wet cool season, and the magnitude of increase of the large follicles was higher (P c C.05) in the dry hot (1.6 + 0.2 to 5.2 + 0.9) than in the wet cool season (0.8 L 0.2 to 3.6 i. 0.7). Season-by-day interactions were neither significant in any of the 3 follicle size classes before and during treatment nor in the size of Fl and F2 follicles before the treatment. DISCUSSION The
time
of
onset
of
superovulatory
estrus
after
the
460
Theriogenology
prostaglandin administration was more variable in the wet cool season than in the dry hot season; however, 2 buffalo did not express estrus symptoms in the dry hot season, although they did ovulate and embryos could be recovered from them. In India and in other countries where the daylight increases with ambient temperature, ovarian activity and expression of estrus were both greater in buffalo in the cooler months. Higher serum prolactin levels were observed in buffalo during the summer months and was thought to be one of the reasons for the poor reproductive performance during these months, presumably due to the ovaries being refractory to the circulating gonadotrophins (17). The lowest sexual activity level was observed from March to July which was also the basis for the broad (3,5,10,29,36), classification of the 2 seasons in our present study. The number of CL as determined at palpation per rectum and by serum progesterone concentrations on Day 6 did not differ between the 2 seasons. It has been reported previously (8.38) that CL are fused or embedded deeply in the ovarian stroma in case of the buffalo and thus only in 25% of the buffalo did per rectum assessment coincided with the actual number of CL observed at slaughter (19). However, in the present study, the number of CL was not ascertained by ultrasonography and the animals were not slaughtered for this purpose. The yield of embryos was higher when the buffalo were superovulated in the dry hot season than in the wet cool season; however, the dry hot season appears to be detrimental to the quality of embryos. This is not likely to be due to the effect of repeated gonadotrophin treatments, since a sufficient rest from breeding was provided to the animals between 2 consecutive It has been reported previously superovulation treatments. (22,24) that with repeated superovulation of high responding buffalo, the responses can be further improved with better hormone preparations. Before the start of superovulation treatment, the recruitment of small and medium size follicles differed between the wet cool and dry hot seasons, with greater numbers of follicles in these classes becoming atretic in the wet cool season. It aonears from the profiles of srowth and resression of the largest *follicle (FI) before supero;ulation treatment that the functional status of the dominant follicle in buffaloes does not differ between the seasons, although the size of the largest follicle alone apparently is not a satisfactory criterion for assessing follicular dominance (I4), as it has been demonstrated that morphologically dominant follicles have different levels of atresia during the growing and regressing phases of development (15). However, such studies have required careful dissection of the ultrasonographically detected dominant follicle in various stages of development for histological and biochemical analysis. The diameter of the second largest (F2) follicle decreased constantly in both seasons. The increase in diameter of the F and decrease in diameter of the F2 follicle occurre a follicle
461
Theriogenology
sooner in the dry hot season than in the wet cool season; however, there was a parallel increase in the difference between the diameters of these (F1 and F2) follicles before the start of superovulation in both the seasons, thereby suggesting the presence of functional dominance in this group of buffalo that large irrespective of season. It has been demonstrated follicles present on the ovaries on Day 7 or Day 9 of the estrous cycle in cattle heifers are functional, since they ovulate following prostaglandin-induced luteolysis (7,20 31). In the present study, 4 of the 5 buffalo were superovulated during the first wave of follicular development. One buffalo was superovulated in the second wave during the wet cool season. However, functional dominance and its effect on superovulation still need to be evaluated in the buffalo. Compared with that of the wet cool season, the dry hot season was marked with a progressive decrease in the number of small follicles, a rapid disappearance of follicles from the medium size class, and a rapid increase in large size follicles (Day 3 of treatment) during the superovulation treatment. This suggests that the process of recruitment and passage of follicles from smaller to larger size classes is better in the dry hot explaining the season than in the wet cool season, possibly better yield of embryos in the dry hot season. However, these embryos were not of transferable quality, and the reason for this could not be ascertained. This study needs to be conducted on a larger group of animals; however, it is difficult to find cyclic during the dry hot buffalo throughout the year, especially months. In cattle more than 80% of the ovulations are reported to occur over a 2-d period in response to superovulation, and it has been suggested that the remaining ovulations represent follicles which could be less mature and which did not respond normally to the LH surge (26,39). In superovulated buffalo the timing of ovulations in response to the LH surge and its effect on oocyte maturation with regard to recovery of good quality embryos is yet to be studied. REFERENCES 1.
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20. Kastelic JP, Knopf L, Ginther OJ. Effect of day of prostaglandin F &;?yP";; o~,s~~e,c,'i~i~dRe~~~~lo~~~n~99~~ the ovulatory 23:169-180. 21. Lindsell CE, Murphy RD, Mapletoft RJ. Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle. Theriogenology 1986; 26:209-219. buffalo: 22. Misra AK. Superovulation and embryo transfer in Progress, problems and future prospects in India. Buffalo J 1993;1:13-24. Kasiraj R, 23. Misra AK, Joshi BV, Agrawala PL, Sivaiah S, RangareddiNS, Siddiqui MU. Multiple ovulation and embryo transfer in Indian buffalo (Bubalus bubalis). Theriogenology 1990;33:1131-1141. 24. Misra AK, Joshi BV, Kasiraj R, Sivaiah S, Ranqareddi NS. superovulatory regimen buffaloImproved for (Bubalus bubalis) . Theriogenology 1991;35:245 abstr. of 25. Moor RM, Kruip AM Th, Green D. Intraovarian control folliculogenesis: Limits to superovulation? Theriogenology 1984;21:103-116. populations during the 26. Pierson RA, Ginther OJ. Follicular estrous cycle in heifers. Anim Reprod Sci 1988;16:81-95. of 27. Progesterone assay. In: WHO Programme for the Provision Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology Method Manual. Geneva, WHO, 1989; 67-79. of 28. Quirk SM, Hickey GJ, Fortune JE. Growth and regression ovarian follicles during the follicular phase of the oestrus cycle in heifers-undergoing spontaneous and PGF2,induced luteolysis. J Reprod Fertil 1986;77:211-219. village herds of Indian water 29. Rao AVN, Rao CS. Oestrus-in buffaloes. Indian Vet J 1970;47:742-748. Keenan L, 30. Savio JD, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J Reprod Fertil 1988;83:663-671. MR, Roche JF. 31. Savio JD, Boland MP, Hynes N, Mattiacci heifers Will the first dominant follicle of the cycle of ovulate following luteolysis on day 7? Theriogenology 1990; 33~677-688. the 32. Sirois J, Fortune JE. Ovarian follicular dynamics during monitored real-time cycle in heifers estrous by ultrasonography. Biol Reprod 1988;39:308-317. 33. Sinsh G, Sinsh GB, Sharma SS, Sharma RD. Studies on oestrous symptoms of buffalo heifers. Theriogenology 1984;21:849-858. follicular on 34. Sinqh G, Singh GB, Sharma ss. Studies patterns in buffalo-heifers. Theriogenology 1984;22:453-462. The Iowa Methods, 35. Snedecor GW, Cochran WG. Statistical State University Press,1967;329-330. and SP, Jain LS, Gupta HK, Bhatia JS. Oestrus 36. Tailor conditions. under village conception rates in buffaloes Indian J Anim Sci 1990;60:1020-1021.
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37. Taneja M. Totev SM. Pawshe CH, Ali A, Sinsh G. Effect of GnRH- incorporation into superovulation regimen for buffalo (Bubalus bubalis) . Proc 12th Intl Congr Anim Reprod, Hague, 1992;1:278-280. Wright Jr RW, Mehmood A, Baig SM. Endocrine 38 Ullah N, profiles in relation to ovarian response, recovery rate and quality of embryos in Nili-Ravi buffaloes treated with FSH. Buffalo J 1992;1:47-56. and onset 39 Yadav MC, Walton JS, Leslie KE. Timing of the duration of ovulation in superovulated beef heifers. Theriogenology 1986;26:509-521.