Use of human intravaginal tampon embedded with natural progesterone induces synchronous estrus in Santa Inês ewes

The production of Santa Inês sheep in Brazil is very important to farmers because they are rustic, adapted to most part of gastrointestinal parasites and have good maternal ability (Sousa et al., 2003; Amarante et al., 2004). To maintain these characteristics, the animals are selected, and reproductive biotechnologies are applied to induce estrus with progestagens throughout the year, due to their seasonality. Different strategies of estrus induction and synchronization are used worldwide (Balaro et al., 2017) but, traditionally, intravaginal progestagen devices are often applied. One of the disadvantages of hormonal treatment is the cost itself. Currently, there are three different commercial brands in Brazil and overall, the cost of those devices is relatively high. Alternatives must be proposed in order to make efficient treatments, without adversely affecting the reproductive efficiency. Therefore, the aim of this study was to evaluate the efficiency of three protocols of synchronous estrus induction during the transition period in Santa Inês ewes. 

The production of Santa Inês sheep in Brazil is very important to farmers because they are rustic, adapted to most part of gastrointestinal parasites and have good maternal ability (Sousa et al., 2003;Amarante et al., 2004). To maintain these characteristics, the animals are selected, and reproductive biotechnologies are applied to induce estrus with progestagens throughout the year, due to their seasonality. Different strategies of estrus induction and synchronization are used worldwide (Balaro et al., 2017) but, traditionally, intravaginal progestagen devices are often applied. One of the disadvantages of hormonal treatment is the cost itself. Currently, there are three different commercial brands in Brazil and overall, the cost of those devices is relatively high. Alternatives must be proposed in order to make efficient treatments, without adversely affecting the reproductive efficiency. Therefore, the aim of this study was to evaluate the efficiency of three protocols of synchronous estrus induction during the transition period in Santa Inês ewes. Blood was sampled by jugular puncture into heparinized vacuolated tubes always in the morning (0600 to 0800h) previously to feeding on Days 0 (D0) (immediately before device insertion), D0+6h (6h after), daily from D1 to D6 (progesterone removal) and 24h after (D7), After collection, the tubes were kept at 5 C until centrifugation at 1,500×g for 15min. The plasma was then aspired and stored at −20 C until analyzed for progesterone (P4) concentrations. The time spent from blood collection to plasma aspiration time did not exceed two hours. Plasma P4 concentration (ng/mL) was determined through the use of the solid phase radioimmunoassay (RIA), using a commercial kit (Beckman Coulter; Immunotech, Marseille, France) at the Hormonal Dosage Laboratory, Animal Reproduction Unit, Fluminense Federal University. The assay sensitivity and intra-assay coefficients of variation were 0.05ng/mL and 12%. In addition, all data were within the maximum and minimum points of the curve.
After device removal, estrus was monitored with the use of fertile rams twice a day (0700 and 1900h) and females were considered to be in estrus when allowed to be mounted. Ewes displaying signs of estrus were mated by fertile Santa Inês rams (n= 4) at the onset of estrus and every 12h if they were still in estrus. Rams were used for approximately equal numbers of ewes from each treatment, with a ram:ewe ratio about ≤ 1:6. Approximately 30 days after mating, conception rate was diagnosed by a B-mode transrectal ultrasonographic scanner (KX2000G Vet ® , Kaixin, Xuzhou, China) with multifrequential (4.5 to 7.5MHz) transducer adapted with a plastic rigid tube for use in small ruminants.
The variables determined were: estrous response (number of females in estrus/number of females induced×100), interval to estrus (h) (interval from device removal to first time ewe stood to be mounted), duration of estrus (interval from the first to last acceptance of mounting), conception rate (number of pregnant females/number of females exposed×100), plasma progesterone concentration (P4; ng/mL). Statistical analysis was performed using all tests with P< 0.05 considered significant. Parametric variables were submitted to one way analysis of variance (ANOVA) and compared by Tukey test by the SAEG program (System for Statistical Analysis). Non-parametric variables were analyzed by the use of the chi-square test. The results are described as mean ± SEM.
Reproductive end points are listed in Table 1. Estrus response rate was overall 87.5% (21/24). In sheep, it is well-known that plasma progesterone concentration (P4) increases from basal levels after ovulation, reaches the plateau during diestrus and becomes progressive lower reaching levels inferior to 1ng/mL during proestrus (Quirke et al., 1979). At this time, this lower level of P4 (<1ng/mL), also called as subluteal level (Minton et al., 1991;Channa et al., 2017) implies in decreasing the negative feedback of progesterone on LH pulse frequency. As a consequence, final follicular development was possible culmination in estrus behaviour and ovulation (Ginther et al., 2006). In the present study it was observed P4 under 1ng/mL (Figure 1).
A total of fourteen ewes (14/24) had supraluteal P4 (>1ng/mL) on Day 0. P4 superior to 1ng/mL at the moment of device removal were detected in all females in Control group, and in five ewes in OB 400 group. All the ewes in OB 200 group, had subluteal P4 (<1ng/mL) at the moment of device removal. P4 was similar (P> 0.05) in all treatments throughout the experimental period, except at the day of device removal, when Control had greater (P< 0.05) P4 (2.5±0.3ng/mL) than OB 200 (0.6±0.1ng/mL) and OB 400 (1.2±0.4ng/mL). On the following day (Day 7) P4 decreased to subluteal concentrations in all ewes of the OB 200 group, and in seven ewes of the Control and OB 400 group of treatments. According to Figure 1, OB 200 ewes showed in average P4 near to limit considered as luteal until Day 4 after device insertion. Interestingly, on Day 5 (24h prior to device removal), all OB 200 ewes showed P4 inferior to 1ng/mL. It could mean that this level of P4 was not capable to inhibit LH efficiently. Thus, possibly this was the reason for shorter interval to estrus in this group. In addition, three ewes from OB 400 group showed P4 inferior to 1ng/mL at device removal. This fact might be involved in intermediary interval to estrus observed in this group.
In the present study, we compared the outcomes of using a human intravaginal tampon embedded with either 200 or 400mg progesterone with the CONTROL group, a commercial progesterone device commonly used for estrus synchronization in sheep. All protocols studied had a satisfactory estrus response and conception rate after the treatments. Although the rates of estrus response and conception were statistically similar, the animals in the OB 200 group had a lower (numerically) response compared to the other two groups. Ewes from OB 400 group showed interval to estrus (31h) similar to CONTROL and also to those previously observed in the breed using estrous induction protocols using 60mg MAP sponges for six days 32h (Cavalcanti et al., 2012).
In conclusion, results of the present study suggest that different intravaginal devices and with different dose of progesterone can be successfully applied for estrus induction in Santa Inês ewes, without adversely affecting reproductive efficiency.