season and that developed a pre-ovulatory wave within an 6 to 98 hour period after male introduction
| Livestock Research for Rural Development 28 (9) 2016 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The influence of separation distance between males and females before breeding season (BS) on reproductive parameters of non-cycling postpartum ewes raised under tropical semi humid conditions was evaluated. Females were evenly distributed into three groups and isolated from males at distances of 1000 m (T1), 300 m (T2) and 3 m (T3) during 30 days before male effect onset.
P4 concentrations (ηg/mL) before BS and on day 9 of BS after male contact were 0.62±0.14/3.35±0.31 (T1), 0.75±0.13/3.75±0.36 (T2) and 0.66±0.22/3.51±0.33 (T3), respectively, which were lower before BS onset. Estrus were detected in 100% females until the 33th day of the BS, while most were detected before the 10th day of the BS. Pre-ovulatory peaks of LH (ηg/mL) of two females of each treatment were evaluated at 42 and 82 (T1), at 22 and 46 (T2) and at 34 and 38 hours (T3) after exposure to males, with mean ovulatory follicle of 7.7±0.5 (T1), 7.5±0.2 (T2) and 7.6±0.4 mm (T3), and mean number of ovulations of 1.5±0.2 (T1), 1.6±0.1 (T2) and 1.4±0.1 (T3). Pregnancy rates were 90.0% (T1), 86.6% (T2) and 90.0 (T3) with prolificacy of 1.30±0.4 (T1), 1.38±0.5 (T2) and 1.33±0.5 (T3). There was no difference between treatments for all evaluated parameters. In conclusion, pre-conditioning distance does not affect male efficiency and further suggests reduction in BS duration to 35 days.
Keywords: biostimulation, fertility, ovis aries, ram effect
Sheep production is an economic activity performed on almost all continents under diverse edaphoclimatic conditions for production of meat, skin, wool and milk, for a wide array of applications. Reproduction plays a major role in productivity in most, if not all, sheep production schemes. Moreover, climatic conditions dictate reproductive outcome to a large degree. The investigation of reproductive technologies under different climatic conditions may help increase sheep production worldwide.
Male effect is an approach having the benefits of easy implementation, low cost and proven efficiency in sheep (Caldas et al 2015), goats (Alves et al 2015; Oliveira et al 2016) and was recently reported in cattle (Silva Filho et al 2015). Besides these advantages, it allows estrous induction without hormone-based protocols, avoiding meat and milk production with residual contamination by exogenous hormones. This fact also complies with animal welfare recommendations and addresses growing concerns by consumers about food production practices (Martin and Kadokawa 2006).
Male effect is defined as a social stimulus (Ramirez and Quintero 2001) capable of triggering cyclicity onset in females under seasonal or postpartum anestrous, by introduction of males into previously isolated flock of ewes at distances of 1000 m or more for several weeks (Pearce and Oldham 1988; Cushwa et al 1992). It was recently reported that, under semiarid conditions, male effect can be applied without any detrimental effect using preconditioning distance of 3 m (Caldas et al 2015). This short distance avoids only physical contact between genders. However, physiology basis for these results remains to be described (De St Jorre et al 2014).
Subtle introduction of males into a female flock promotes an increase in LH pulse release and reduces the E2 negative feedback over the hypothalamic-pituitary axis that ultimately results in a LH pre-ovulatory wave (Martin et al 1986). Moreover, increase in LH pulse frequency stimulates follicular development, E2 secretion and ultimately ovulation (Perkins and Fitzgerald 1994).
Progesterone (P4) reduces the LH pre-ovulatory wave, a fact that may influence oocyte maturation and follicle luteinization. Moreover, first ovulation in ewes under anestrus and subjected to male effect may lead to low fertility rates, due to low LH levels during the luteal phase. This fact can only be overcome by exogenous P4 usage concomitant with male effect (Skinner et al 2000).
Based on these facts, the influence of pre-conditioning between males and females under distances of 1000 m, 300 m and 3 m upon cyclicity of Santa Inês ewes under tropical semi humid conditions was evaluated. Also to evaluate the LH pre-ovulatory peak, P4 concentrations before and after BS, ovulatory follicle diameter growth, pregnancy rates and prolificacy.
The experiment was conducted accordingly to ethical standards followed by Brazilian institutions. The project was formally approved by an ethics committee from the Veterinary College at Pio Décimo, Aracaju, Sergipe state, Brazil (Protocol n.08/2012). The work was conducted in Escada, Pernambuco state, Brazil. The geographic coordinates are latitude 08° 21' 33'' S, longitude 35° 13' 25'' O, altitude de 109 m, mean annual temperature of 24.4 °C and mean annual rainfall of 1763 ml. The weather is tropical semi humid, with rainfall from May to August.
Animals were raised in pastures during the day and returned to pens during the afternoon. Animals were fed with cultivated (Brachiaria humidicola) and native pastures (Paspalum maritimum, Chloris orthonton, Cynodon dactylon, Brachiaria tunnergrass), and further supplemented with hay ( Pennisetum purpureum) when kept in the pen. Animals had free access to mineral salt and water (ad libitum). Main sanitary practices were removal of manure from pens once a week and systematic deworming and vaccination against rabies and clostridiosis.
Females with age from 2 to 4 years that had delivered within 15 to 30 days were initially evaluated for body score condition, where females with a score of 2 to 3 (Gonzalez-Stagnaro 1993). Furthermore, females were also pre-selected using ultrasonography of the reproductive tract to determine the cyclicity status.
Ultrasound exams were performed by the same technician throughout the experiment using an Aquila Pro equipment with 6.0 and 8.0 MHz linear transducer adapted using a PVC support in order to facilitate the manipulation in the rectum of the animal. Pre-selected females were identified with numbered plastic ear tags, and were maintained in isolation from males for 30 days in order to avoid any physical, visual, olfactive and auditive contact within the two experimental groups (T1 and T2), separated by 1000 m and 300 m respectively. Separation of genders by 3 m (T3) only avoided physical contact between them.
One week before initiation of the experiment, blood samples of all ewes were collected by jugular vein puncture in vaccutainer tubes containing heparin. Blood serum was stored at -20 ˚C, until P4 concentration analysis by radioimmunoassay. Females were scored as non-cycling when P4 concentration were lower than 1 ηg/mL (Morales et al 2003). After P4 concentration analysis, females (n = 90) were randomly and equitably allocated to experimental groups.
Santa Inês rams (n = 3) were selected accordingly to their reproductive performance (proven fertility) and submitted to an andrology exam one week before experiment. Bucks were marked with a mixture of grease and ink (4:1) around the sternum bone region, in order to facilitate identification of cycling females before being introduced into groups. Rams were marked with inks of different colors and were exchanged between groups on days 10, 20 and 30 of the BS.
After the introduction of males into female flocks, three females from of each group were randomly subjected to blood collection during a 98 hour period with four hour intervals. This approach was used to determine luteinizing hormone (LH) concentrations. Blood serum was also used for determination of LH by radioimmunoassay. The increase in LH concentration was considered significant when it exceeded over 20 ηg/mL, for two consecutive measurements (Martin et al 1983). Concentration of P4 was measured on the 9th day after BS, and ewes were scored as cycling when displayed P4 concentration of 1 ηg/mL or higher.
Ovarian activity was evaluated daily by ultrasonography after estrus detection in six females of each experimental group. Ultrasonography was performed by same technician, who also counted and measured growing antral follicles. The moment of ovulation was defined as the time point when the ovulating follicle disappeared, followed by corpus luteum formation at the same location on the same ovary (Tenório Filho et al 2007).
Estrus was observed twice a day (6:00 and 16:00 hours) by trained personnel during a BS of 45 days, and estrus was considered synchronized when detected within the first initial five days of the BS. Pregnancy diagnosis was performed by ultrasonography on 35th and confirmed on the 60th day after the last mating.
Parametric variables were submitted to analysis of variance and compared by the SNK test in System for Statistical Analysis (SAEG) software, with results being presented as means and standard deviation. Means were compared by Unpaired T test. Non-parametric variables were evaluated by the chi-square test, and were presented as percentages. A 5% difference between groups was considered significant.
Table 1 shows the data on P4 blood concentration. P4 concentrations were lower than 1 ηg/mL, indicating that all females were non-cycling before exposure to males. On the same table, it is possible to verify that after the introduction of males, females cycled and ovulated, displaying differences in P4 concentrations (P < 0.05).
|
Table 1. P4 blood concentration of non-cycling Santa Inês ewes, that had delivered within 45 and 60 days, before and after the 9 th day of male contact and that were previously isolated from males during 30 days for 1000 m (T1), 300 m (T2) and 3 m (T3) before onset of an 45 day-breeding season. |
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|
Treatment |
N° of |
P4 Concentration (ηg/mL) |
p |
|
|
Before |
After |
|||
|
T1 |
30 |
0.62±0.14a |
3.35±0.31b |
< 0.0001 |
|
T2 |
30 |
0.75±0.13a |
3.73±0.36b |
< 0.0001 |
|
T3 |
30 |
0.66±0.22a |
3.51±0.33b |
< 0.0001 |
|
Different superscript letters on same line differ at p < 0.05. |
||||
Figure 1 displays LH blood concentration after introduction of males into females flocks. It became evident that, irrespectively of the pre-conditioning distance, male effect induced LH pre-ovulatory peak between 38 and 62 hours after resuming breeding season.
|
| Figure 1.
LH Concentration (ηg/mL) in Santa Inês ewes, that had delivered within
45 and 60 days, that were previously isolated from males for 30 days for
1000 m (T1), 300 m (T2) and 3 m (T3) before the onset of 45-day breading season and that developed a pre-ovulatory wave within an 6 to 98 hour period after male introduction |
Figure 2 illustrates estrus dispersion that was observed until day 33 of the BS. Estrus detection was observed throughout the BS, where most estrus where detected within the initial ten days. First estrus detection for female was from day 1until day 33 of the BS, and mean first estrus in days was 15.5 ± 10.4 (T1), 9.25 ± 6.41 (T2) and 13.1 ± 10.2 (T3) days. It is also interesting to note on that estrous synchronization (Figure 2), or estrous detected within the initial five days of the BS reached 23% for all females, 26% for T1, 20% for T2 and 23% for T3, with no difference (P > 0.05) between groups.
 |
| Figure 2. Estrus detection in Santa Inês ewes, that delivered within 45 to 60 days, that were kept apart from male for 30 days for distances of 1000 m (T1), 300 m (T2) and 3 m (T3) before onset of the breeding season of 45 days |
Table 2 contains information on single and double estrous, as well as ovulatory follicles diameter and total number of ovulations. It can also be observed that all females displayed at least one estrous, and single and double estrus were observed at similar rates (P > 0.05). It can be observed that ovulatory follicle diameter and number of ovulations did not differ (P > 0.05) between groups (Table 2).
|
Table 2. Percentage of single (one) and double estrus, as well as mean values (± s) of diameter (mm) of ovulatory follicle and number of ovulations in Santa Inês ewes, that delivered within 45 to 60 days, that had previously from males for 30 days for 1,000 m (T1), 300 m (T2) and 3 m (T3) before a breeding season of 45 days. |
|||||
|
Treatment |
Estrus |
Ovulation |
|||
|
Simples |
Double |
Total |
Follicle |
Total |
|
|
T1 |
30 |
0.62±0.14a |
3.35±0.31b |
< 0.0001 |
1.5±0.2 |
|
T2 |
30 |
0.75±0.13a |
3.73±0.36b |
< 0.0001 |
1.6±0.1 |
|
T3 |
30 |
0.66±0.22a |
3.51±0.33b |
< 0.0001 |
1.4±0.1 |
|
Different superscript letters on same line differ at p < 0.05. |
|||||
Table 3 contains data regarding pregnancy on first and second services, conception rates, as well as prolificacy for all groups. No difference was observed between groups for these criteria (P > 0.05).
|
Table 3. Percentage de pregnancy per number of services of Santa Inês females, that delivered between 45 and 60 days, that were previously isolated from males during 30 days for 1,000 m (T1), 300 m (T2) and 3 m (T3) before breeding season for 45 days. |
|||
|
Treatment |
Pregnancy per Service |
||
|
First |
Second |
Total |
|
|
T1 |
18/27 (66.6)a |
9/27 (33.4)b |
27/30 (90.0) |
|
T2 |
19/26 (73.0)a |
7/26 (27.0)b |
26/30 (86.6) |
|
T3 |
18/27 (66.6)a |
9/27 (33.4)b |
27/30 (90.0) |
|
Different superscript letters on same line differ at p < 0.05. |
|||
Table 4 shows that all pregnant females delivered singletons or twins. The percentage of singletons deliveries were substantially higher (P < 0.05) than twin deliveries. However, prolificacy did not differ (P > 0.05) between groups.
|
Table 4. Delivery type and prolificacy of Santa Inês ewes that had delivered within 45 to 60 days, and isolated from males for 30 days at 1,000 m (T1), 300 m (T2) and 3 m (T3) before a breeding season of 45 days |
|||
|
Treatment |
Delivery type |
Prolificacy |
|
|
Singletons |
Twins |
||
|
T1 |
19/27 (70.4)a |
8/27 (29.6)b |
1.30±0.4 |
|
T2 |
18/26 (69.2)a |
8/26 (30.8)b |
1.38±0.5 |
|
T3 |
18/27 (66.7)a |
9/27 (33.3)b |
1.33±0.5 |
|
Different superscript letters on same line differ at p < 0.05) |
|||
Mean P4 concentrations immediately before introduction of males into female flocks were in accordance with conditions described for non-cycling females (Morales et al 2003). Mean values on day 9 after BS were in agreement with cyclicity status, demonstrating male effect potential for inducing fertile estrus in ewes under postpartum anestrus.
Ovulations described here were due to LH ovulatory peaks that occurred in a moderately dispersed time frame, independent of preconditioning distance and BS onset. It was initially hypothesized that longer distances would increase ovulation rates. However, it is important to note that an immediate increase in pulse frequency and ultimately LH concentration was observed after male effect. Other authors have also reported the role of male effect on LH pulses and its concentration increase (Martin et al 1983; Atkinson and Williamson 1985; Minton et al 1991).
As described here, LH peaks were observed within the initial 42 hours of interaction between genders. High ovulation rates within the initial 72 hours after exposure to males was previously reported and were attributed to increased LH basal concentrations (Oldham et al 1979). However, all females described here were responsive to male effect despite their anestrus condition. Therefore, these females are very unlikely to have displayed high LH basal concentrations before BS onset. This hypothesis is in disagreement with a report that higher LH basal concentrations immediately before contact with males, which contributed to increased sensibility of females to the male effect (Martin et al 1980). Since LH concentrations were not evaluated before BS onset, it may suggested that the increase in LH concentration was due to male exposure, and its high variability between measurements as described here, possibly due to LH low stability or half-life.
Estrous was observed until day 33 of the BS. It was possible to note that during this period of time, females with two estrous cycles were similar to those that displayed one estrous cycle and based on these findings, collectively with the high percentage of synchronized estrous, it is possible to suggest a reduction in breading season duration from 45 to 35 days. This reduction in BS duration would result in lower production costs, especially specialized labor, further further concentrating lambing during more favorable periods of the year. This would lead to more uniform lamb lots and further allow ovine production in accordance to market demands (Fonseca 2005). It is still important to note that the number of ovulations was similar across all preconditioning groups. This shows that stimuli provided by males was efficient despite distances between genders, at least for a preconditioning period of 30 days. Using wool breeds, some authors described ovulatory follicles varying from 5.1 to 5.7 mm (Guinther et al 1995; Evans et al 2001). Ovulatory follicles with greater diameters could be due to male effect, which led to increased FSH secretion and greater E2 production. Similar results were also observed in Nellore cows subjected to biostimulation (Silva Filho et al 2015).
Pregnancy rates were in agreement with other reports under semiarid conditions (Caldas et al 2015; Tenório Filho et al 2016). Pregnancy rate for first service were higher than for second service, irrespectively of preconditioning distance. These findings do not agree with independent observations that first estrous after male effect are anovulatory (Ungerfeld et al 2004). Alternatively, if ovulation occurs, corpus luteum may be weak, regress faster and would lead to estrous cycles of shorter duration (Chemineau et al 2006). Estrus induction was observed in most females, a fact that demanded intense sexual activity from males within initial ten days of the BS. This fact could have contributed to similar pregnancy rates between services, or more efficiency on second service since fewer estrus were detected around this period. Sufficient forage disposability may have minimized effects of energetic deficit caused by postpartum condition. Moreover, usage of males with proven fertility and under adequate male to female ratio rules out some putative confounding factors on ewe’s reproductive outcome (Alves et al 2014).
Deliveries showed a significant prevalence of singletons over twins, a fact that was previously described elsewhere (Mexia et al 2004). Prolificacy is a trait under more intense influence of breed-specific factors. As described here, it was similar to data described for Santa Inês and Morada Nova ewes (Machado et al 1999; Caldas et al 2015).
Male effect induces and synchronizes estrus of postpartum ewes, without affecting pregnancy rates and prolificacy, irrespectively of preconditioning distance between males and females.
Moreover, these findings further suggests that breeding season duration could be shortened to 35 days, since females that did not conceive on the first service could have a second chance to conceive during the same breeding season.
The authors are grateful to CNPq and CAPES for financial support of this study.
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Received 7 June 2016; Accepted 7 August 2016; Published 1 September 2016