Gerardo Duarte

EVIDENCE THAT MELATONIN ACTS IN THE PREMAMMILLARY HYPOTHALAMIC AREA TO CONTROL REPRODUCTION IN THE EWE : PRESENCE OF BINDING SITES AND STIMULATION OF LUTEINIZING HORMONE SECRETION BY IN SITU MICROIMPLANT DELIVERY

Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.

Reproductive seasonality in female goats adapted to a subtropical environment persists independently of food availability.

The aim of this study was to determine whether the seasonal change in food availability provides a predictive cue that synchronizes the breeding season in goats adapted to the subtropical conditions of Northern Mexico. Groups of multiparous intact (n=7) and ovariectomized does bearing a subcutaneous implant constantly releasing estradiol-l7 beta (OVX+E; n=12) were allocated in two pens and received a constant amount of feed. Another group of OVX+E does (n=10) was incorporated into a flock raised under natural grazing conditions with seasonal fluctuations in food availability. Blood samples were taken twice weekly. Ovulation of intact goats was inferred from plasma progesterone levels. In OVX+E does plasma levels of LH were measured. Intact does displayed seasonal variations in ovulation and the breeding season lasted from September to February. OVX+E goats displayed large seasonal variations in LH secretion regardless of whether they were fed constantly indoors or kept under natural grazing conditions (P<0.0001). The mean date of increase in LH secretion varied between years (P<0.0001) but did not differ between groups (naturally grazed: August 13+/-10 days; constantly fed: August 26+/-10 days). In contrast, the seasonal decline in LH secretion differed between groups: it was consistently delayed in constantly fed animals compared to those feeding naturally (naturally grazed: January 28+/-4 days; constantly fed: February 24+/-6 days, group effect, P<0.01). These results show that local female goats from subtropical Mexico display large reproductive seasonality independently of food availability.

Male effect in seasonally anovulatory lactating goats depends on the presence of sexually active bucks, but not estrous females

A study was conducted in subtropical northern Mexico (26 degrees N) to determine whether the presence of estrous females can improve the response of seasonally anovulatory goats to the introduction of bucks in the group. The induction of estrous activity was studied in three groups of anovulatory lactating goats during seasonal anestrus. These females were of the Mexican Creole breed. In the control group (sexually inactive (SI), n = 20), two control (SI) bucks exposed to normal seasonal daylength variations were used. In the second group (SI + E, n = 20 + 3), two control males were also used, but in addition, three females of the group were in estrus at the time of male introduction. In the third group (sexually active, SA + E, n = 19 + 4), anovulatory females were exposed to two bucks made sexually active by exposure to 2.5 months of long days (16L:8D) followed by two subcutaneous 18 mg melatonin implants, and four estrous females were also present when introducing the bucks. In all groups, males were introduced on 15 March and estrous detection was conducted twice daily for 15 days. The sexual activity of the bucks was observed from 08:00 to 10:00 h during the first five days of exposure to females. More females displayed estrous behavior in the first 15 days following the introduction of the males in the SA + E group (18/19) as compared with the SI or SI + E groups (2/20 and 0/20, respectively; P < 0.001). No difference was observed between the two latter groups. Thirteen females of SA + E group showed a second estrus between days 6 and 11 (short estrous cycle duration: 5.4 +/- 0.4 days). By contrast, in the SI group none showed a second estrus. The sexual behavior of the males in the SA + E group was greater as compared with that of the males in SI and SI + E groups (over 80% of the total sexual activity recorded in the three groups; P < 0.001). By contrast, no differences were found between SI and SI + E males. These results indicate that the presence of estrous females alone at the time of buck introduction is not sufficient to induce an adequate stimulation of seasonally inactive males. The use of sexually active bucks is necessary to induce reproductive activity in anovulatory females, whereas preparation of the bucks with long days followed by melatonin implants allows them to gain such a capacity.

Ovulatory activity of female goats adapted to the subtropics is responsive to photoperiod

The objective of this study was to determine whether reproductive seasonality of local well-fed female goats from subtropical Mexico (26 degrees N) can be controlled by photoperiod. The control group (n=12) remained in an open pen under natural photoperiod variations. The two experimental groups (n=8 each) were placed in light-proof rooms and exposed for 2 yr to alternations of 3 months of long days and 3 months of short days. One group was first exposed to long days, Group 1, and the other one to short days, Group 2. Blood samples were obtained twice a week to determine ovulation status by progesterone plasma concentrations. Goats from the control group displayed a seasonality of ovulations. The mean (+/-SEM) dates of the onset and end of the ovulations were September 10+/-5 d and February 16+/-4 d, respectively. In contrast, in both experimental groups, ovulations were modified (P<0.05) by the photoperiodic treatments such that ovulations started and ended during short and long days, respectively. In Groups 1 and 2, ovulations started 67+/-2 and 66+/-2 d (P>0.05), respectively, after the transfer from long to short days. In contrast, the timing of the cessation of ovulations after the transfer from short to long days differed (P<0.05) between groups (19+/-3 and 31+/-3 d for Groups 1 and 2, respectively). Local female goats from subtropical Mexico are, therefore, sensitive to photoperiodic changes that they are exposed to at this latitude and this environmental cue may control the timing of the breeding season in natural conditions.

Male goat vocalizations stimulate the estrous behavior and LH secretion in anestrous goats that have been previously exposed to bucks.

We investigated whether live vocalizations emitted by bucks interacting with anestrous females stimulate secretion of LH, estrous behavior and ovulation in anestrous goats. In experiment 1, bucks rendered sexually active by exposure to long days followed by natural photoperiod were exposed in a light-proof-building to five anestrous females. Buck vocalizations were reproduced through a microphone-amplifier-loudspeaker system to an open pen where one group of goats (n=6) was exposed for 10 days to these live vocalizations. Another group of females (n=6) was isolated from males and vocalizations. The proportion of goats displaying estrous behavior was significantly higher in females exposed to buck vocalizations than in females isolated from males. The proportion of goats that ovulated did not differ between the 2 groups (exposed to males versus isolated). In experiment 2, female goats that either had previous contact with males (n=7), or no previous contact with males (n=7) were exposed to live buck vocalizations, reproduced as described in experiment 1, for 5 days. The number and amplitude of LH pulses did not differ between groups before exposition to buck vocalizations. Five days of exposure to male vocalizations significantly increased LH pulsatility only in females that had previous contact with males, while LH pulse amplitude was not modified. We concluded that live buck vocalizations can stimulate estrous behavior and LH secretion in goats if they have had previous contact with bucks.

Importance of intense male sexual behavior for inducing the preovulatory LH surge and ovulation in seasonally anovulatory female goats

The present study was carried out to determine whether the presence of photostimulated sedated male goats could stimulate the LH preovulatory surge and ovulation in seasonal anestrous goats. Sexually experienced male goats were treated with artificial long days (16 hours light per day) from 1 November to 15 January to stimulate their sexual activity in March and April, corresponding to the natural sexual rest. A female group of goats (n=20) was exposed to non-sedated males who displayed an intense sexual behavior and provided strong odor (non-sedated group). Another female group of goats (n=20) was exposed to the photo-stimulated male goats, but these males were sedated with Xylazine 2% to prevent the expression of sexual behavior (sedated group). The sedated males also provided a strong odor. Females of both groups had full physical and visual contact with non-sedated or sedated males. In both groups, the males remained with females during 4 days. The LH preovulatory surge of 10 female goats per group was measured by determination of LH plasma concentrations in samples taken every 3 hours. In addition, in all goats, (n=20 by group), ovulation was determined by measuring plasma concentrations of progesterone. The proportion of female goats showing a preovulatory LH surge was higher in goats exposed to non-sedated (10/10) than in those exposed to sedated bucks (0/10; P<0.0001). Similarly, most of does in contact with non-sedated males ovulated (19/20), but none of those in contact with sedated males did so (0/20; P<0.0001). We conclude that the expression of an intense sexual behavior by male goats is necessary to induce LH preovulatory surge and ovulation in seasonally anovulatory goats.

Sexually active bucks are able to stimulate three successive groups of females per day with a 4-hour period of contact

Bucks rendered sexually active by a photoperiod treatment of long days can induce fertile ovulation in a group of goats with only 4 h of contact daily with a male:female ratio of 1:10. Here we tested whether such bucks could induce fertile ovulations when stimulating successively three different groups of anovulatory goats when interacting 4 h per day during 15 consecutive days. Control males (n=3) were introduced in the control group (n=25) of does at 8:00 h and were removed at 12:00 h. Experimental males (n=3) were in contact with the experimental groups of does: from 8:00 h to 12:00 h with a first group (n=27), from 12:00 h to 16:00 h with a second group (n=26) and with a third one (n=27) from 16:00 h to 20:00 h. Bucks were then placed until next day in another pen. Both in the control and the experimental groups, more than 85% of females ovulated, and the proportions did not differ between the control and experimental groups (P≥0.67) or between the three experimental groups (P≥0.67). Moreover, the ovulation rate did not differ significantly between the control and the experimental females nor between the three experimental groups. Bucks were able to fertilize more than 72% of does independently of the number of females they were exposed to (P≥0.17). Finally, more than 58% of females kidded and fertility did not differ between the control and experimental groups (P=1) nor among experimental groups (P≥0.77). We conclude that sexually active bucks are able to induce fertile ovulation in three successive groups of anovulatory goats even when the period of contact between sexes is reduced to 4 h per day.

Lack of sexual experience does not reduce the responses of LH, estrus or fertility in anestrous goats exposed to sexually active males.

We investigated whether LH secretion, estrous behavior and fertility would differ between sexually inexperienced and experienced anestrous goats exposed to the males. Male goats were rendered sexually active during the reproductive rest season by exposure to 2.5 months of artificial long days. Two groups of anovulatory sexually inexperienced and sexually experienced does were exposed to males during 15 days (n = 20 per group). LH pulsatility was determined every 15 min from 4h before to 8h after introducing males (Day 0). Estrous behavior was recorded twice daily. Pregnancy rates were determined on Day 50. Fertility was determined at parturition. Male sexual behavior was registered on days 1 and 2 during 1h. Before introducing the males, the number of LH pulses did not differ between groups. After introduction of the males, all females increased their LH pulsatility, but the number of pulses did not differ between sexually inexperienced and experienced goats. The proportion of females displaying estrous behavior with a high pregnancy rate and fertility did not differ between inexperienced and experienced goats. The sexual behavior of the males did not differ significantly between those interacting with sexually inexperienced or experienced goats. We conclude that goats can show substantial endocrine and reproductive responses to males, even in the absence of previous sexual experience, when sexually active bucks are used.

Maintaining bucks over 35 days after a male effect improves pregnancy rate in goats

The ovulation can be induced by the introduction of a male among a group of anovulatory goats, a phenomenon called the ‘male effect’. The objective of this study was to determine whether maintaining bucks over 35 days after the initial introduction of males, improves pregnancy rate in goats. We used sexually active bucks, which were joined (n = 2 each) with three groups of goats (n = 30 each): (i) males remained with females for 15 days (Control group); (ii) males remained with females for 35 days (same buck group); (iii) males were removed at Day 23 and replaced by other males, which were not familiar to the females and which remained with females until Day 35 (new buck group). Percentage of goats ovulating did not differ among groups at Day 19 (≥87%; P = 0.12). Pregnancy rates at Day 35 did not differ between groups (≥70%; P = 0.90). At Day 55, pregnancy rates were similar in the same and new buck groups (96%; P = 1.0), but were higher than that of the Control group (73%; P < 0.05). Therefore, maintaining bucks over 35 days after the onset of the male effect improved pregnancy rate more than 20 points compared with females that remained with the same bucks for 15 days only.

Absence of previous sexual experience did not modify the response of anoestrous goats to photo-stimulated bucks in Spring

Abstract Sexually inexperienced rams display lower frequency of nudging, ano-genital sniffing, mounting attempts, and mounts with intromission than experienced ones. Experienced photo-stimulated males display high level of sexual behaviour. The aim of this study was to determine: i) whether sexually inexperienced photo-stimulated males display similar sexual behaviour as the experienced ones, and ii) whether these inexperienced bucks can stimulate sexual response in anoestrous goats, as the experienced ones. Sexually inexperienced and experienced photo-stimulated males (n = 5 each) were used. In April, two groups of anovulatory females (n = 50 each) were exposed to these males for 15 d. Male sexual behaviour was registered for three days, upon their introduction into female groups. On day 1, the inexperienced males displayed higher nudging than the experienced ones (p < .001), whereas on days 2 and 3, the experienced males displayed higher nudging than the inexperienced ones (p < .001). The ano-genital sniffing, mounting attempts, and mounts with intromission did not differ between the inexperienced and experienced groups (p > .05). The proportion of females ovulating did not differ between the two groups (≥82 %; p = 1.0). Pregnancy rate (≥68 %; p = .829) did not differ between the females exposed to inexperienced or experienced males. It is concluded that sexually inexperienced photo-stimulated males show similar sexual behaviour as the experienced ones, and they are able to induce sexual response in seasonally anoestrous goats through the male effect.