Early development and location of embryos in the reproductive tract of Nili Ravi buffalo (Bubalus bubalis): a retrospective analysis

One year data on embryo recovery were analyzed to study the development and descent of preimplantation embryos in Nili Ravi water buffalo. Forty-five superovulatory attempts were performed on 23 buffalo. A total of 45 embryos were recovered either nonsurgically or after slaughtering the animals at various time intervals (85 to 176 h) post estrus. Embryos were located in the oviducts at 85 h after estrus. At 108 h post estrus, most of them (78%) were recovered from the uteri. The embryos had 8 to 16 cells at 85 h post estrus, grew to morulae at 108 h and to compact morulae at 125 h post estrus. Early blastocysts were observed at 141 h post estrus. Blastocysts were predominant (69%) at 156 to 176 h after estrus; no hatched blastocysts were recovered during this time interval. Based on our findings, embryo recovery at around 150 h post estrus (i.e., Day 6 of the cycle) is recommended for compact morulae or blastocysts in the water buffalo.     

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.     

Successful nonsurgical embryo transfer in buffalo (Bubalus bubalis ) in Bulgaria

Forty-one Day 5.0 to Day 5.5 embryos and one unfertilized ovum were recovered nonsurgically from 24 superovulated, parous buffalo (Bubalus bubalis ) and transferred nonsurgically to 28 synchronized recipients by a team of Bulgarian and American scientists. Five pregnancies were established and four live buffalo calves were born at the end of normal gestation periods.     

Bubaline versus bovine reproduction

Fertility in water buffalo (Bubalus bubalis) is considerably lower than that in cattle (Bos taurus and Bos indicus). Poor breeding efficiency is attributed to late onset of puberty, seasonality, poor estrus expression, and long calving intervals. Accurate estrus detection is a prerequisite for efficient reproductive management. Established reproductive management techniques in cattle can be successfully applied to water buffalo because of the similarities in the anatomy, physiology, and endocrinology of reproduction between the two genera.     

Induction of ovulatory estrus and fertility in non-cycling buffaloes with norgestomet during low breeding season

Field trials were designed to evaluate use of norgestomet treatment to induce ovulatory estrus in non-cycling buffalo cows and heifers during low breeding season. Twenty-five buffalo cows and 50 heifers under village management were given a 9-day treatment with a polymer implant containing 6 mg norgestomet with IM injections of 5 mg estradiol valerate + 3 mg norgestomet at the time of implantation and 600 IU PMSG when the implant was removed. Fifty animals served as controls without any treatment. Seventy-four treated animals showed estrus during the period between 36 to 80 hours after removal of the implant. Twenty-five buffalo cows and 40 heifers that could be further followed up were inseminated twice at 8-hour intervals, 12 hrs after induction of estrus with chilled semen by recto-vaginal method. Of these, 15 (23.1%) conceived, 9 (36%) among buffalo cows and 6 (15%) among heifers. Fourteen buffalo cows and 30 heifers that did not conceive manifested cyclic estrus at an interval of 22.4 and 20.6 days. The conception rate in the cyclic estrus was 57% and 23.3%, respectively, for buffalo cows and heifers. The overall conception rate over two inseminations was 46.2%, 68% in buffalo cows and 32.5% in heifers. In the control group, five (10%) showed spontaneous estrus and two (40%) conceived during the period of the experiment.     

Monitoring ovarian cyclicity in postpartum Murrah buffalo through milk progesterone enzyme immunoassay

Milk samples were collected from Murrah buffalo between Day 30 and Day 120 post partum and analysed for progesterone concentration to monitor ovarian cyclicity. Progesterone levels were low (1 to 5 ng/ml) during the anestrous period. Levels were also low around estrus, but they began to increase at Day 6 postestrus; high levels (15 to 32 ng/ml) were maintained for different periods. There was a marked drop in progesterone level after Day 16 to 18 of the estrous cycle in those animals which returned to estrus. Progesterone levels remained high in buffalo which did not return to estrus, indicating that these animals were pregnant. Some of the progesterone cycles were not associated with the expression of estrus. This study indicated that a milk progesterone enzymoimmunoassay can be used to detect early pregnancy as well as conditions such as silent estrus and anovulatory estrus; it can thus help reduce the long intercalving period in buffalo.     

Superovulatory response of river bufalo (Bubalus bubalis )

The ovarian response of 25 buffalo-cows was visually assessed, and their oviducts and uteri separately flushed 3 to 6 d post superovulatory estrus at slaughter. Ten buffalo-cows slaughtered on Days 5 and 6 were examined per rectum for corpora lutea (CL) and follicles > 8 mm prior to slaughter, and the estimate was compared later with the actual ovarian response. Five out of the ten buffalo-cows were nonsurgically flushed in vivo on Day 5 of the estrous cycle, a day before slaughtering, and as a result, six ova/embryos were recovered. After the flushing of the reproductive tract at slaughter, one more ovum was recovered from the uterus of each of the three buffalo-cows. As a result of treatment of three groups of five buffalo with 3000 IU pregnant mare serum gonadotrophin (PMSG) on Days 6, 10 or 14 of the estrous cycle, 3.8, 6.2 and 3.4 CL on the average were recovered, respectively (Experiment I). A mean number of 8.8 and 9.0 CL, respectively, was obtained in two groups of five buffalo each, after treatment with 40 mg of follicle stimulating hormone (FSH) on Day 10 of the stage of the estrous cycle (Experiment II) and 3000 IU PMSG regardless of the stage of cycle (Experiment III). The percentage of ova/embryos recovered in the three experiments was 32.8, 20.4 and 22.2, respectively.     

Assessment of superovulatory responses in terms of palpable corpora lutea and embryo recovery using milk progesterone

Milk progesterone profiles were used to assess superovulatory responses in cyclic lactating buffalo (n = 9) in terms of the number of ovulations and the number of embryos recovered. All of the buffalo received a total of 30 ml of folltropin divided into morning and evening doses and spread over 5 days, beginning on Day 10 of the estrous cycle (day of expected estrus = Day 0). Milk samples for progesterone determination were collected on alternate days from all nine animals from Day 1 prior to the expected synchronized estrus to 5 days after flushing for embryo recovery. All animals were palpated per rectum 1 day prior to flushing in order to record the number of corpora lutea. Of an estimated 23 ovulations from the nine buffalo, only 12 embryos were recovered, of which one was an unfertilized oocyte. Milk progesterone profiles from individual buffalo suggested that a poor superovulatory response in terms of embryo recovery in some buffalo was caused by a failure to respond optimally to lutalyse treatment for the induction of estrus. It was hypothesized that ova trapping by the fimbriae of the fallopian tubes may not be efficent in this species especially in the superovulated ovaries.     

In vitro fertilization of water buffalo follicular oocytes and their ability to cleave in vitro

Water buffalo (Murrah) oocytes were collected from ovaries obtained from a local slaughterhouse. They were classified according to the character of the cumulus cells under a stereomicroscope and then cultured in 25 mM Hepes buffered tissue culture medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO(2) in air at 39 degrees C. After 20 to 24 hours of in vitro maturation, the oocytes were cultured at 38.5 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO(2) in air. Oocytes with compact and dense cumulus cells cleaved significantly further (P<0.01, 67.3%, 33 49 ) than those with fair, partially denuded oocytes with thin cumulus layers (27.5%, 25 91 ) or small remnants of cumulus cells and poor naked oocytes (3 100 ). A substantial variation in fertilization and developmental rates (16.0 to 43.8%) was observed among 4 different bulls. Late morulae were transferred nonsurgically into 14 buffalo recipients on Day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by palpation per rectum on Day 60 and delivered a calf in October 1991.     

Gas chromatographic-mass spectrometric analysis of chemical volatiles in buffalo (Bubalus bubalis) urine

Isolation of active fraction and characterization of chemosignals from urine have been attempted in several mammalian species in the recent years. The objective of this study was to identify the urinary volatiles across various reproductive stages of buffalo cow, namely, estrus, diestrus, and pregnancy, and in bull, by chemical extraction followed by gas chromatography–linked mass spectrometry (GC-MS). Urine samples were collected from six buffalo cows at two different phases of estrous cycle, namely, estrus and diestrus. Besides, urinary samples were collected from five pregnant buffalo cows (60–75 days after artificial insemination (AI)) and six adult bulls. Thin-layer chromatography was performed as a preliminary test for qualitative comparison of different compounds extracted by organic solvents. Identification of the urinary compounds was carried out in a gas chromatograph (Perkin Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass). The results of GC-MS analysis indicated the presence of 21 compounds with varying molecular weights and retention time, which were further categorized as diestrus-specific, pregnancy-specific, and bull-specific urinary compounds. No compound, however, could be identified as estrus-specific. We concluded that qualitative differences do exist in estrus, diestrus, and pregnant buffalo cow urine and in bull urine, as evidenced by GC-MS.     

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.     

Estrus synchronization with lower dose of PGF2 alpha and subsequent fertility in subestrous buffalo

Two experiments were conducted to determine luteal regression, estrous response and fertility in buffalo receiving cloprostenol via 2 routes of administration. In Experiment 1, cyclic buffalo (n = 10) were assigned to 2 equal groups receiving either 500 micrograms i.m. cloprostenol (Estrumate, ICI) or 125 micrograms cloprostenol injected intravulvosubmucosal (ivsm) ipsilateral to the side of the corpus luteum (CL) on Day 11 of an induced estrous cycle. Serum progesterone (P4) concentrations were evaluated immediately before treatment and at 24, 48, 72, 96 and 120 h after PGF2 alpha administration. The decline in serum P4 concentrations was significantly different (P < 0.05) between groups up to 48 hrs after treatment. However, no significant difference (P > 0.05) was observed for the interval from treatment to the onset of estrus (94.9 +/- 10.7 vs 96.0 +/- 15.9 h) for 500 or 125 micrograms of cloprostenol groups, respectively. In Experiment 2, multiparous, lactating subestrous buffaloes (n = 137) were treated either with 125 micrograms ivsm cloprostenol or 500 micrograms i.m. cloprostenol (n = 28 vs 33, respectively) during peak breeding (September-February) or low breeding (March-August) season (n = 37 vs 39, respectively). Buffalo observed in estrus were inseminated twice with frozen-thawed semen at 12 and 22 h after the onset of estrus. Buffalo that failed to exhibit estrus were given a second equal dose of cloprostenol at an 11-d interval and underwent fixed-time insemination at 72 and 96 h. The interval to the onset of estrus was 85.0 +/- 4.4 vs 73.2 +/- 2.6 h during peak breeding and 96.1 +/- 2.6 vs 92.1 +/- 3.8 h during the low breeding season for buffalo treated with 125 and 500 micrograms cloprostenol, respectively. These intervals were different (P < 0.05) between seasons but not between treatments in the same season. Conception rates of 47.8 vs 53.1% during peak breeding and 23.5 vs 25.6% during low breeding season were also different (P < 0.05) between seasons but not between the treatments in the same season for buffalo treated with 125 and 500 micrograms cloprostenol, respectively. These results indicated that 125 micrograms ivsm and 500 micrograms i.m. cloprostenol were equally effective for synchronizing estrus in subestrous buffalo. No negative effect of a lower dose of cloprostenol was observed on estrus synchrony and subsequent fertility; however, season of treatment had a significant effect on conception rates.     

Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus)

Information on the use of buffalo follicular fluid (buFF) in modulation of ovarian functions in farm animals is scanty compared to other species. This is an attempt to investigate the effect of direct administration and active immunization of 30 kDa and above buFF proteins on ovarian functions in goats. Treatment of goats (n = 6) with steroid free 30 kDa and above buFF protein fraction during late-luteal phase for 4 days (days 12 or 13 to days 15 or 16) of the natural cycle, delayed the onset of estrus by 24 h compared to control although the mean duration of estrus was unaffected. A 71% increase (P = 0.06) in mean ovulation number was also observed following treatment. However, the population of large (> or =5 mm diameter) follicle was not affected. The ovarian activity calculated as total of ovulation and large follicles increased (1.6 times) significantly (P = 0.02) in treated animals. Active immunization of goats (n = 5) against these proteins did not affect the onset and duration of estrus. Similarly, the ovulation rate, number of large follicles and the ovarian activity did not differ significantly between immunized and control groups. The study revealed that 30 kDa and above buffalo follicular fluid contains some factor(s) that cause delay in the onset of estrus in goats and increase the ovulation rate. Active immunization against these proteins in goat did not show any effect either on onset, duration of estrus or ovulation rate and large follicle population. Detailed study on these buffalo follicular fluid proteins may help to use them further for modulation of ovarian function in farm animals.     

Peripheral inhibin levels during estrous cycle in buffalo (Bubalus bubalis)

A sensitive, specific RIA was validated and used for measurement of peripheral plasma immunoreactive inhibin (irinhibin) levels during the estrous cycle in Murrah buffalo. The RIA employed an 125-I iodinated inhibin as tracer and an antiserum against dimeric inhibin. The procedure had a sensitivity of 16 pg/tube, and the nonspecific effects of buffalo plasma were compensated for by including 200 ul bullock plasma in the standards. Separation of free and bound inhibin was affected by the use of a second antibody and precipitation with polyethylene glycol. Blood samples were collected once daily for 30 d from Murrah buffalo (n = 6) during the hot month of July. Cyclic activity and estrus were confirmed by plasma progesterone determination. Peripheral plasma concentrations of ir-inhibin fluctuated between 0.40 +/- 0.07 and 0.67 +/- 0.13 ng/ml during the estrous cycle in buffalo. During the same period, plasma progesterone levels increased from 0.21 +/- 0.01 ng/ml at Day 0 to a peak of 3.30 +/- 0.72 ng/ml on Day 13, declining sharply by Day -5. Ir-inhibin levels exhibited an increase during the follicular phase, with the maximum concentration of 0.65 +/- 0.01 ng/ml occuring on the day of estrus, a decline thereafter, and no pattern during the luteal phase. The differences, however, were not statistically significant throughout the estrous cycle.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Bull-specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis)

This study was conducted to compare fertilization rate and viable embryo recovery rate in superovulated buffalo (n = 64) following insemination with semen from buffalo bulls (n = 5) having different fertility rates as determined by AI. Frozen-thawed semen from fertile bulls with similar post-thaw progressive motility and sperm morphology was used to inseminate buffalo at superovulatory estrus. Fertilization and viable embryo recovery rates differed among bulls, but this bull-specific effect was not related to the overall herd fertility rate as determined by AI in normal cyclic animals. These results indicate that individual bulls differ in their contribution to fertilization of superovulated donors and also to embryonic development, as determined by viable embryo recovery. Moreover, the results also suggest that buffalo bulls can be screened for optimal fertility and embryo recovery rates in superovulated donors. Further studies are warranted to ascertain the factors which contribute to such bull-specific effects.     

Effect of prostaglandin F(2alpha)- thamsalt and Estrumate (ICI 80996) on plasma progesterone levels in Indian water buffaloes (Bubalus bubalis )

Thirty six buffalo showing normal reproductive cyclas were given a single intramuscular injection of prostaglandin F(2alpha)- thamsalt (PGF(2alpha)) and Estrumate on day 9 or 15 of the estrous cycle in order to induce estrus. In PGF(2alpha) treated animals, plasma progesterone dropped from 2.99+/-0.29 to 0.10 +/- 0.02 ng/ml and from 5.53 +/- 0.53 to 0.09 +/- 0.03 ng/ml (mean +/- SEM) within 72 hours of treatment, on day 9 and 15, respectively. Similarly, the treatment with Estrumate on day 9 or 15 of the cycle reduced the level of proges--terone from 3.02 +/- 0.03 to 0.14 +/- 0.03 ng/ml and from 4.26 +/- 0.47 to 0.21 +/- 0.03 ng/ml, respectively, 72 hours aPter each treatment. It was inferred that both drugs induce estrus in buffalo without any observed residual effect.     

Effect of CRESTAR on estrus synchronization and the relationship between fecal and plasma concentrations of progestagens in buffalo cows

In buffaloes estrus synchronization provides an opportunity for enhanced use of AI; however, changes in hormone secretion during synchronization are poorly understood. The aim of this investigation was to determine if the concentration of progesterone metabolites in feces would correlate with the concentration of progesterone in blood and thus, could be used for noninvasive monitoring of the reproductive status in buffalo cows. Additionally, the influence of a norgestomet-estradiol treatment (CRESTAR-ear implant) was investigated. According to the clinical examination and the progesterone profile in blood samples during the three wks before the treatment, the 17 animals were allotted to 3 groups: 1) CL = presence of corpus luteum throughout the period of 3 wks before the treatment (n = 8); 2) CY = cyclic, corpus luteum present for less than 3 wks (n = 6); and 3) AE = anestrous, with inactive ovaries (n = 3). In the first group, 4 animals started an estrous cycle after implant withdrawal and conceived after natural mating. In the second group one of the cyclic cows showed estrus two d after implant withdrawal, the other 3 had a delayed estrus (12 to 16 d). The two cows which had had inactive ovaries at the beginning but were cyclic before the treatment started, remained cyclic after implant withdrawal but did not become pregnant. The 3 anestrous cows of the third group remained anestrous after the treatment. The progesterone concentration in blood clearly correlated with the concentration of the metabolites in feces. Therefore, this noninvasive method is a valuable tool for determining the luteal status, and such information may be useful for developing estrus synchronization regimens in buffalo cows.     

Rate of transport and development of preimplantation embryo in the superovulated buffalo (Bubalus bubalis)

This study was designed to ascertain the rate of transport and development of preimplantation embryo in the superovulated buffalo in order to determine the optimum time for their nonsurgical collection. Eighteen Murrah-type buffalo were superovulated with 600 mg NIH-FSH-P1. Luteolysis was induced by administration of PGF2 alpha at 72 (PG + 72) and 84 h (PG + 84) after initiating gonadotrophin treatment and fixed-time AI was done beginning at 36 h post PG + 72 administration and at 12-h intervals thereafter, upto 72 h. Six control buffalo received treatment similar to experimental group except that in place of FSH they received normal saline. For embryo collection, experimental animals were humanely killed at 6-h intervals corresponding to 156 (n = 2), 162 (n = 2), 168 (n = 2), 174 (n = 3), 180 (n = 3), 186 (n = 3) and 192 h(n = 3) after PG + 72 treatment, whereas the control animals were humanely killed at 156 (n = 2), 174 (n = 2) and 192 h (n = 2). Superovulated buffalo had higher number of ovulations than untreated controls (8.78 +/- 5.00 vs 0.67 +/- 0.51) and total ova/embryos recovered was 4.11 +/- 2.46 and 0.67 +/- 0.51, respectively. The high estradiol-17 beta (E2) levels with its prolonged rise may, by leading to reverse peristalsis in the oviduct with a consequent loss of some embryos in the peritoneal cavity, be one of the reasons for our inability to recover nearly 84/158 ova/embryos in the superovulated buffalo. In superovulated animals, nearly all the ova/embryos reached the uterus between 168 and 174 h post PG + 72 treatment or about 134 h (circa 5.5 d) after the onset of superovulatory estrus, suggesting that the ideal time for non-surgical embryo collection in the buffalo is between Days 7 to 8 after PG + 72 treatment or Days 5.5 to 6.0 of the superovulated cycle (estrus = Day 0). Embryo development of superovulated buffalo showed considerable variation as various stages of embryos (8 cell to expanded blastocyst) were recovered from the same donor buffalo, and the rate of development appeared to be 24 to 36 h faster than in cattle.