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Estrus detection by using vaginal cytologic examination in miniature swine.

Vaginal smears were obtained from four Yucatan miniature swine daily for 69 days and stained with hematologic stain. Epithelial cells were categorized as superficial, large intermediate, small intermediate, or parabasal. Leukocytes were also quantitated. External signs of estrus were recorded, including swelling, discharge, restlessness, or vocalization. Mean age of three of the swine was 147 days at the beginning of the study. The fourth pig was 317 days old. The three younger swine had their first observed estrus at the age of 178 days (range, 167 to 196 days). Mean cycle length was 17 to 21 days. The moving mean leukocyte count (i.e., each value was averaged with the values for the previous day and the following day) always exceeded the epithelial cell count (regardless of type), except during the 3 to 4 days when the pigs exhibited external signs of estrus. Further, epithelial cells were at their peak during estrus, decreasing markedly during diestrus, and increasing again during proestrus. The combined superficial plus large intermediate cell counts were significantly higher during estrus than during diestrus or proestrus. We conclude that daily vaginal smears can be used to determine the stage of estrus in Yucatan pigs.

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The influence of estrus cycle on neurosteroid potency at the gamma-aminobutyric acidA receptor complex.

The reduced metabolites of progesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one or 3 alpha,5 alpha-P) and deoxycorticosterone (3 alpha,21-dihydroxy-5 alpha-pregnan-20-one or 5 alpha-THDOC) exhibit a gamma-aminobutyric acid (GABA) agonist pharmacological profile and bind with high affinity to a unique steroid recognition site on the GABAA receptor complex (GRC). In female rats, peak levels of 3 alpha,5 alpha-P occur between proestrus and estrus and at concentrations which appear sufficient to affect GRC-mediated inhibitory events. Therefore, steroid modulation of the binding of [35S]t-butylbicyclophosphorothionate was conducted to examine sensitivity of the GRC during the estrus cycle. The results in unwashed tissue (cortex, cerebellum, hippocampus and striatum) indicated that 3 alpha, 5 alpha-P was most potent in estrus. Subsequent studies in washed tissue and in washed tissue plus 3 microM (+)bicuculline indicated that 3 alpha,5 alpha-P was more potent in diestrus 1 than in estrus. Brain regional differences in potency of 3 alpha,5 alpha-P were also observed. Overall, the results in washed tissue suggest that an inherent change in the sensitivity of the GRC occurs during the estrus cycle, whereas the results in unwashed tissue may be influenced by the presence of endogenous GRC-active neurosteroids and GABA. The change in sensitivity of the GRC during the estrus cycle may occur to help maintain homeostatic regulation of brain excitability.

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[Use of PGF2alpha and GnRH for estrus synchronization in cattle].

Estrus detection has a tremendous impact on the reproductive efficiency in dairy farms. Recently the systematic use of hormones for synchronization of estrus and ovulation has been propagated. These programs are designed to facilitate estrus detection and/or increase its efficiency. Prostaglandin programs are used to improve estrus detection and reproductive management in dairy operations. All cows are treated up to three times in weekly or biweekly intervals at the end of the voluntary waiting period. This should lead to groups of cows in estrus within two to four days after PG treatment. Since PG programs cannot completely eliminate the need for estrus detection procedures for fixed time artificial insemination (AI) were developed. Fixed time AI not only requires control of the luteal phase of the cow but also synchronization of the follicular development. Treatment with GnRH will induce a new follicular wave and provide a dominant follicle in a defined growth phase at the time of PG administration seven days later. A second injection of GnRH given 48 hours after PG results in ovulation approximately 24 to 32 hours after GnRH (OvSynch-procedure). The pros and cons of hormone programs in dairy farming are discussed in the light of field trials and experiments performed in our department.

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Estrus and ovulation in gilts fed a synthetic progestogen.

A synthetic progestogen, allyl trenbolone (AT), was fed to sexually mature gilts to determine the effective doses for the control of estrus and ovulation. Gilts were assigned to a control group and 5 treatment groups receiving 10.0, 12.5, 15.0, 17.5 or 20.0 mg of AT mixed in .45 kg of feed/head/day for 18 consecutive days. Ovarian morphology was determined by laparotomy following estrus or at 10 days post-treatment. AT suppressed estrus in all gilts during treatment. Estrus was effectively synchronized in treated gilts. The average interval from withdrawal of progestogen treatment to estrus was 4.5 days for 48 of 50 treated gilts that were in estrus within 10 days after treatment. The average ovulation rate in treated gilts was similar to control gilts. No detrimental side effects, due to treatment, were observed with the possible exception of a slight increase in the incidence of cystic follicles.

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Estrus synchronization and pregnancy rates in cyclic and noncyclic beef cows and heifers treated with syncro-mate B or Norgestomet and Alfaprostol.

Postpartum lactating cows (N=118) and virgin heifers (N=60) were treated with subcutaneous Norgestomet implants for nine days and received either an intramuscular injection (im) of 5 mg estradiol valerate and 3 mg Norgestomet at the time of implant insertion or an im injection of 5 mg Alfaprostol 24 hr before implant removal. Animals were artificially inseminated 12 hr after detection of estrus. Of the cows and heifers, 78% and 88%, respectively, were in estrus within five days after implant removal (P<0.09). There was no difference between treatments in the proportion of animals in estrus or in the timing of estrus (P<0.85). Estrus was detected in a greater (P<0.05) proportion of animals that were cyclic prior to treatment (88%) than among those that were anestrous prior to treatment (77%). Pregnancy rates after five days were similar between heifers that were cyclic (42%) or anestrous (47%) prior to treatment; however, the five-day pregnancy rate in cows that were anestrous prior to treatment was 38% lower than that in cows that were cyclic prior to treatment (17 vs 55%, P<0.01). Although the treatments synchronized or induced estrus in both cyclic and anestrous animals, marked variability in estrous response and fertility among previously cyclic or anestrous postpartum cows limited the effectiveness of the treatments.

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Synchronization of estrus and fertility in goats with norgestomet ear implants.

Estrus was synchronized in 64 dairy goats in July with norgestomet ear implants. Half the does received ear implants that contained 6 mg norgestomet and the remaining does received implants that contained 3 mg. Implants were left in place for 11 days. Each doe received i.m. injections of 400 IU PMSG and 50 mug cloprostenol 24 hours prior to implant removal. Twenty-eight of 32 does (87.5%) that received 6 mg or 3 mg norgestomet exhibited onset of estrus within 24 hours of implant removal. All does had exhibited onset of standing estrus by 43 hours after implant removal. Does were hand-mated to fertile bucks twice daily while in standing estrus. There were no differences between does implanted with 6 mg or 3 mg in fertility to the induced estrus (74.2% vs 75% kidding), mean length of gestation (151.0 +/- 3.2 vs 151.6 +/- 2.0 days), mean number of kids per doe (2.1 +/- 0.8 vs 2.3 +/- 0.7) or in mean kid weights (3.10 +/- 0.80 vs 3.06 +/- 0.86 kg) (6 mg vs 3 mg, respectively). It was concluded that ear implants that contained 3 mg of norgestomet were equally as effective as implants that contained 6 mg for synchronization of estrus in dairy goats.

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The use of heat detection aids in estrus synchronization programs.

The use of tailhead paste and chin-ball markers for estrus detection in beef cows following estrus synchronization was evaluated. The estrous cycles of 85 cows were synchronized with a prostaglandin analogue in a two-injection program. At the time of the second injection, each cow's tailhead was painted with an estrus detection paste and the cows were placed in pastures with four intact bulls with chin-ball markers. Blood samples for plasma progesterone assays were obtained at the time of each injection and daily for five days following the second injection. Based on progesterone values, tailhead paste was incorrect 27% of the time in indicating estrus and chin-ball markers were incorrect 24% of the time. Detecting particular cows in estrus in groups of synchronized cows is difficult even with estrus detection aids.

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Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle.

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48 h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96 h, and sometimes at 120 h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24 h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48 h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.

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Estrogen-induced pseudopregnancy in gilts: Its use in estrus synchronization and subsequent influence on litter response.

The effectiveness of using estrogen-induced pseudopregnancy followed by PGF(2alpha) (Lutalyse) treatment to synchronize estrus in gilts and the subsequent effect of pseudopregnancy on litter parameters was examined in two experiments. Experiment 1 consisted of a control (n = 49) group and a pseudopregnant (n = 49) group. Pseudopregnant gilts received PGF(2alpha) between Days 25 and 38 and were bred at the ensuing estrus. Ninety-two percent of the gilts responded to PGF(2alpha) and exhibited estrus in 4.9 +/- 0.2 (Least Square Mean +/- SEM) days. Duration of estrus was longer in pseudopregnant compared to control gilts (2.8 vs 1.7 d, P<0.001). Experiment 2 consisted of a control (n = 24) group and a pseudopregnant (n = 24) group. All pseudopregnant gilts received PGF(2alpha) on Day 38 and 79% responded with a mean onset of estrus at 5.2 +/- 0.2 d. As in Experiment 1, duration of estrus was longer in gilts from the pseudopregnant group (2.2 vs 1.7 d, P<0.005). The percentage of farrowing was similar for both groups in both experiments, and no differences existed in the number of pigs born and those alive at 21 d post partum. Litters from gilts in the pseudopregnant groups were heavier at birth than those in the control group (Experiment 1, 17.21 vs 15.25 kg, P<0.01; Experiment 2, 15.31 vs 13.55 kg, P<0.02) and at 21 d (Experiment 1, 60.81 vs 56.79 kg, P<0.01; Experiment 2, 51.89 vs 46.96 kg, P<0.02). Survival rate at 21 d was higher in offspring from pseudopregnant gilts in both experiments (Experiment 1, 85.3% vs 78.2%, Experiment 2, 91.4% vs 81.2%).

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An electromyographic study of uterine activity during normal and progestogen-regulated estrus in the ewe.

Myometrial electrography was performed by means of chronically implanted electrodes in the base, the middle and the tubal end of both the uterine horns of six Serres ewes at normal and progestogen-regulated estrus. During the first 24 h of normal estrus, myometrial electrical activity was characterized by intermittent spike bursts (Pattern I activity), occurring at about the same frequency in the three sites of both horns (middle of the right horn: 44.13+/-6.07/30 min). This pattern was then gradually transformed so that 36 h after the beginning of estrus, there was mostly only episodic activity consisting of 18.22+/-3.44 bursts, lasting 6.50+/-2.42 mins, and recurring at 30.51+/-16.24 min intervals simultaneously in both horns (Pattern II activity). During Patterns I and II, the respective ratios of descendingly to ascendingly propagated bursts were 2.65 and 0.78. Pattern I activity also occurred during the first 24 h of progestogen-regulated estrus, but the frequency of the bursts of spikes although occurring in the tubal ends of both horns at about the same rate as in Pattern I at normal estrus, gradually decreased towards the uterine body, (tubal end: 44.34+/-4.28, middle: 40.14+/-5.42, and base: 33.22+/-4.82/30 min). Then Pattern I activity was transformed into a miscellaneous pattern instead of into Pattern II. In the progestogen-regulated estrus, the ratio of the descending to the ascending propagations of spikes during Pattern I activity rose to 5.11.

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Estrus induction in the bitch using a combination diethylstilbestrol and FSH-P.

The purpose of the study was to induce estrus and ovulation in normal bitches using a combination of diethylstilbestrol (DES) and follicle stimulating hormone of porcine pituitary origin (FSH-P). Thirteen mature mongrel female dogs were divided into two groups, the first group was treated for estrus induction during late anestrus and the second group during mid-anestrus. The dogs were monitored by teasing, vaginal cytology, and hormonal assay during the induced (n=13) and the previous spontaneous estrous cycle (n=9). Six of eight and three of five bitches came into standing estrus in the first and second group, respectively. Of the bitches that came into estrus, three conceived in the first group and one in the second. The average induced litter size was 7.0 versus 7.5 for the colony. Based on vaginal cytology the induced proestrus and estrus lasted 1.7 (0 to 3) and 12.9 (4 to 24) d, respectively, while the spontaneous proestrus and estrus lasted 5.8 (0-17) and 12.8 (9-15) d, respectively. Progesterone profiles were similar between the induced and spontaneous estrous cycles, although the progesterone peak was higher during the spontaneous cycle. The preovulatory luteinizing hormone (LH) surge was observed in only one induced estrous cycle. Modest results were obtained with this therapy. However, the litter sizes were normal and the induced cycles were very similar to the physiologic ones. No side effects were seen with the oral form of DES.

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Simultaneous injection of PGF2alpha and GnRH into diestrous dairy cows delays return to estrus.

Simultaneous injections of prostaglandin F2alpha (PGF) and gonadotropin releasing hormone (GnRH) or saline were given to 32 diestrous dairy cows to test the ability of GnRH to improve estrous and ovulation synchrony beyond that of PGF alone. Cows were randomly assigned to receive PGF on Day 8 or Day 10 of the estrous cycle (estrus = Day 0), and all cows were further assigned to simultaneous injection of GnRH or saline. Corpus luteum (CL) regression, return to estrus and follicular activity were monitored by plasma progesterone assay, twice-daily estrous detection and ultrasonographic examination, respectively. Plasma progesterone concentrations declined to <1.0 ng/ml at 24 hours after PGF in all cows and were not affected by GnRH. Gonadotropin releasing hormone inducted premature ovulation or delayed return to estrus in 7 of 8 cows treated with PGF/GnRH on Day 8 and 3 of 8 cows treated with PGF/GnRH on Day 10. Further, cows with premature GnRH-induced ovulations failed to develop and maintain a fully functional CL, and all returned to estrus 7 to 13 days after the induced ovulation. These data indicate that GnRH administered simultaneously with a luteolytic dose of PGF disrupts follicular dynamics and induces premature ovulation or delays normal return to estrus and, therefore, does not improve the synchrony of estrus and ovulation achieved with PGF alone.

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Synchronization of estrus in suckled postpartum beef cows with melengestrol acetate and PGF2alpha.

A total of 402 two suckled postpartum beef cows at 2 locations (CSU and ECRC) were utilized in 3 trials to evaluate the effectiveness of a combination melengestrol acetate (MGA)-PGF2alpha estrus synchronization system in spring-calving cows. The cows were allocated by days post partum, body condition score (1 = emaciated; 9 = obese), sire breed, and dam age at the beginning of treatment to 1 of 2 treatments within location and trial: MGA-PGF2alpha (0.5 mg MGA/head/d for 14 d with 25 mg of PGF2alpha injected 17 d after MGA withdrawal), and unsynchronized controls. All cows were observed for estrus at 12-h intervals for at least 5 d post injection. Cows observed in standing estrus were inseminated 12 to 18 h later. There was a location effect on response to treatment that was attributed to differences in body condition score between locations so data were analyzed within a location. Body condition score at the CSU location was 5.7 compared with 4.0 at the ECRC location. The CSU MGA-PGF2alpha treated cows had higher (P < 0.05) 5-day estrus and 5-d pregnancy rates (78.6 and 61.0%, respectively) than the CSU controls (11.1 and 6.9%, respectively). Similar results for 5-d estrus and pregnancy rates but of decreased magnitude were also observed for the ECRC MGA-PGF2alpha treated (31.6 and 21.4%, respectively) cows compared with that of the ECRC controls (11.9 and 8.5%, respectively). The CSU MGA-PGF2alpha treated cows had higher (P < 0.05) 25- and 60-d pregnancy rates (82.5 and 94.8%) than the CSU controls (65.3 and 87.5%). The 25- and 60-d pregnancy rates were similar between the ECRC MGA-PGF2alpha treated cows and ECRC control cows. The MGA-PGF2alpha estrus synchronization system appears to contribute to pregnancy early in the breeding season in postpartum beef cows, although its effectiveness is limited by cow body condition.

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Synchronization of estrus in suckled postpartum beef cows with melengestrol acetate, 48-hour calf removal and PGF2alpha.

One hundred and sixty-five suckled postpartum beef cows were utilized to evaluate the effectiveness of 2 estrus synchronization systems for the initiation and synchronization of estrus. The treatment groups consisted of 1) melengestrol acetate (MGA)-PGF2alpha (cows were given 0.5 mg MGA/head/day for 14 d with 25 mg PGF2alpha injected 17 d after the last day of MGA administration); 2) MGA-48-h calf removal (CR)-PGF2alpha (cows were given 0.5 mg MGA/head/day for 14 d with 48-h calf removal starting on the second day after completion of the MGA regimen plus 25 mg PGF2alpha administered 17 d after the last day of MGA); and 3) unsynchronized controls. Cows were assigned to treatments by the numbers of days post partum, body condition, age, and breed of sire. The cows were observed for estrus at 12-h intervals for 5 d after PGF2alpha administration and were artificially inseminated 12 to 18 h after the observed estrus. Both the MGA-PGF2alpha and MGA-CR-PGF2alpha treatments (64.8 and 61.8%) had greater (P < 0.05) 5-d estrus rates than the control treatments (34.5%). The synchronized pregnancy rate was greater (P < 0.05) for the MGA-CR-PGF2alpha than the control treatment.(52.7 vs 30.9%, respectively). The MGA-CR-PGF2alpha cows had a higher 25-d pregnancy rate than either the MGA-PGF2alpha (P < 0.05) or control cows (P < 0.08). Of the anestrous cows at the beginning of treatment, more MGA-CR-PGF2alpha (P = 0.1) and MGA-PGF2alpha cows were cyclic posttreatment than control cows (58.7 and 55.1 vs 44.7%, respectively), suggesting that treatment initiated estrous cycles in only a small number of the anestrous cows. Both MGA-PGF2alpha and MGA-CR-PGF2alpha treatments appear to be effective methods of synchronizing estrus in suckled postpartum beef cows. However, MGA-CR-PGF2alpha was more effective in establishing pregnancy earlier in the breeding season than MGA-PGF2alpha.

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Comparison of two MGA-PGF2alpha systems for synchronization of estrus in beef heifers.

Yearling beef heifers (n=193) were used to evaluate reproductive performance attained with 2 MGA-PGF2alpha synchronization systems. These treatments were compared with an untreated control group. The 14-d MGA heifers were synchronized by feeding 0.5 mg MGA/h/d for 14 d. At 17 d after the last MGA feeding, these heifers were injected with PGF2alpha (25 mg, im). Heifers in the 7-d MGA treatment group were fed 0.5 mg MGA/h/d for 7 d and received a 25-mg, im injection of PGF2alpha on the last day of the MGA feeding period. Heifers in all 3 treatment groups were observed for estrus every 12 h for 7 d beginning 24 h after the PGF2alpha injection. Heifers observed in estrus during this 7-d period were artificially inseminated approximately 12 h after the onset of estrus. The percentages of heifers in estrus during the 7-d synchronized period were 75.4, 56.3 and 17.2% for the 14-d MGA, 7-d MGA and control groups, respectively. The estrous responses were significantly different in each treatment. The percentage of heifers in estrus during the peak 24-h period was higher (P<0.05) in heifers synchronized with the 14-d MGA system than in heifers synchronized with the 7-d MGA system (75.5 vs 50.0%). The synchronized conception rate of the 14-d MGA heifers was significantly higher (65.3%) than that of both the 7-d MGA (41.7%) and control (45.4%) heifers. Synchronized conception rates were similar (P=0.79) in the 7-d MGA and control treatments. Synchronized pregnancy rates were 55.2, 32.4 and 15.2% for the 14-d MGA, 7-d MGA and control groups, respectively. Both synchronization treatments resulted in significantly higher synchronized pregnancy rates compared with that of the controls. The synchronized pregnancy rate was higher (P<0.05) in the 14-d MGA group than it was in the 7-d MGA group. The mean day of conception within the breeding season was 11.5 and 9.3 d shorter in the 14-d MGA heifers than in the 7-d MGA and control heifers, respectively. Our results indicate that using the 14-d MGA system to synchronize estrus in beef heifers results in better reproductive performance than that attained in heifers synchronized with the 7-d MGA system or in control heifers.

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Effects of different stimuli of service on estrus duration in dairy goats.

The object of this experiment was to study the effects of different stimuli of service on estrus duration in dairy goats. Twenty Nubian goats were assigned randomly to 4 groups of 5 animals each: service (SER), mechanical stimulation of vagina (MES), accessory gland fluid insemination (AGF), and control (CON), Estrus was synchronized by using medroxyprogesterone acetate intravaginal pessaries (60 mg) over a 12-d period. Estrus was detected using 1 aproned vasectomized buck at 6-hour intervals during 5 d after pessary removal (at 0600, 1200, 1800 and 2400 h). In the SER group the male was permitted to service each female. In the MES group, stimulation was accomplished using a penis-like device maintained in the vagina 15 sec with light pressure on the fornix. In the AGF group, 1.0 ml of accessory gland fluid was deposited into the external cervical os. The CON group was only permitted to be mounted. All treatments were performed only once within the first 12 h of estrus. Estrus duration for the SER, MES, AGF and CON groups was (mean +/- SD) 22.8 +/- 5.0, 27.6 +/- 6.8, 37.2 +/- 2.7 and 42.0 +/- 9.5 h, respectively. The SER group was different from the AGF and CON groups (P<0.01), but not from the MES group (P>0.05). The MES group was different from the AGF (P<0.05) and CON groups (P<0.01). The AGF and CON groups did not differ from each other (P>0.05). It is concluded that service shortened estrus duration due to the mechanical effect of stimulation of the penis-like device against the vaginal fornix.

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Effect of service and vaginal-cervical anesthesia on estrus duration in dairy goats.

The object of this experiment was to study the effect of sterile service and vaginal and cervix anesthesia on estrus duration in dairy goats. During the fall season 21 Nubian goats (9 nulliparous and 12 multiparous) were randomly assigned to 1 of 3 treatment groups (n = 7 animals per group). The following groups were formed: service (SER), vaginal and cervix anesthesia (VCA) and control (CON). Estrus was synchronized using fluorgestone acetate intravaginal pessaries (FGA, 30 mg) over a 14-d period. Estrus was detected using a vasectomized buck at 6-h intervals over 5 d after pessary removal (at 0600, 1200, 1800 and 2400 h). In the SER group the male was permitted to service each female. In the VCA group the vagina and cervix of the does were anesthesied, after which the male was permitted to service the females. Both treatments were done once within the first 12-h initiation of estrus. Does were permitted to be mounted only in the control group (CON). Estrus duration for SER, VCA and CON groups was (mean +/- SD) 24.0 +/- 10.9, 42.0 +/- 15.9 and 40.3 +/- 10.8 h, respectively. The SER group was significantly different from the VCA and CON groups (P < 0.01); however, the VCA group was not different from the CON group (P > 0.05). It is concluded that service shortens the duration of estrus due to the mechanical effect of the penis against the vagina and cervix, and not to the accessory gland fluid.

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Occurrence of flehmen in male buffaloes (Bubalus bubalis) with special reference to estrus.

Pheromonal cues present in the urine and vaginal mucus during the estrus period elicit courtship behaviour in mammals including the characteristic flehmen behaviour. In the present study, the flehmen behaviour was assessed in male buffaloes by exposing them to heifers for three consecutive estrous cycles. Behavioural observations revealed that the bulls sniffed the female's external genitalia, responded to the chemical signal(s) and exhibited the flehmen behaviour similar to those reported in other ungulates. The flehmen behaviour was recorded daily during 15-min contact with females for three consecutive estrous cycles. Out of 180 observations during five days estrus periods (-3 to +1 days) which included high frequency of flehmen behaviour and out of 637 observations with or without flehmen behaviour during diestrus periods, the bull displayed 365 and 441 flehmen behaviour, respectively. Average numbers of all (2.03 +/- 0.66) and repeated flehmen (1.05 +/- 0.64) behaviour during estrus periods were significantly higher than those of diestrus periods of all (0.69 +/- 0.25) and repeated flehmen (0.11 +/- 0.10). The statistical significance was higher (P < 0.001) in repeated flehmen behaviour of estrus as compared to that in diestrus. In case of all flehmen behaviour, the statistical significance was higher (P < 0.01) in estrus when compared to that in diestrus. The occurrence of flehmen behaviour in male buffaloes clearly indicates that the specific pheromonal compound(s) present in the urine/vaginal mucus during estrus significantly influence the flehmen behaviour.

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