The Efficacy of Three Different Estrus Synchronization Protocols on Reproductive Performance in Chinese Hu Sheep

Sheep is one of the most vital components of agribusiness economy of China. The superiority of Hu sheep, a non-seasonal breeder raised in Jiangsu and Zhejiang Province of China, is that its onset of estrus and gestation event works out in all seasons and multiparous trait (Yue, 1996), which makes it a desirable alternative for increasing reproductive performance. Significant improvement in reproductive performance can be accomplished via application of exogenous hormones including gonadotropins, progestogens and luteolytic agents to induce secretion of endogenous hormones (Oliveira et al., 2001; Abecia et al., 2012; Menchaca et al., 2018). The critical theory of such methods is to manipulate luteolysis and the corpus luteum lifespan (Titi et al., 2010). The purpose of application of estrous synchronization protocols is to control the reproductive system, which has become a worldwide available solution for modern management and large-scale cultivation of sheep (Boscos et al., 2002). Additionally, compared with natural mating, artificial insemination could rise the numbers of descendant per ram and allow a spatial and temporal (in term of frozen-thawed semen) separation between collection of spermatozoa and fertilization, as well as saving time (Leboeuf et al., 2000; De et al., 2015). The application of CIDR dispenser and/or injection of exogenous hormones, prostaglandin F_2α (PGF_2α), equine chorionic gonadotropin (eCG), was considered as a section of the protocol (Nogueira et al., 2011; Kulaksiz et al., 2013). Another protocol for controlling the estrus cycle is based on the lysis of the corpus luteum using PGF_2α (Fierro et al., 2013). PGF_2α treatments have the advantage of being applied by intramuscular injection and be rapidly and almost completely (99%) metabolized by the liver, which was always combined with GnRH agonist (Hashem et al., 2015). It has been reported that the implement of intravaginal devices may induce several problems, such as the production of chemical residues and vaginitis that can threaten to public health (Alavez-Ramirez et al.,  2016).
       
Applicable synchronization protocol could significantly change the onset and interval and duration of estrus as well as the rates of pregnancy and lambing, thus increasing productivity and fertility of livestock (Kulaksiz et al., 2013). It has been reported that CIDR-based estrous synchronization protocols in goats have included short (5-9 days) (Oliveira et al., 2001; Nogueira et al., 2011; Inya et al., 2013) and long duration (12-14 days) (Motlomelo et al., 2002; Romano, 2004; Abecia et al., 2012) with the combination applications of PGF_2α and eCG at or prior to insertion removal. Promising results were obtained by application of progestogen for 9 days (Nogueira et al., 2011), 8 (Greyling et al., 1979), 7 (Fitzgerald et al., 1985) or 5 (Beck et al., 1993) in combination with PGF_2α, or the analogue cloprostenol, at withdrawal time. Martinez et al., (2018) observed that the ewes received the long-term CIDR-based treatment for 14 days could lead to higher fertility yields (70%) compared with that of long-term sponge-based treatment for 14 days (45%). Ataman et al., (2006) found that ewes received intramuscular injection of GnRH analogue and treatment of PGF_2α analogue 5 day later were proved to be more effective in the synchronization of the estrus than those received given twice PGF_2α​ at 9 day interval, intramuscular injection of PGF_2α. However, the comparisons in the efficacy of CIDR-long treatment, CIDR-short treatment and GnRH-PGF_2α treatment to synchronize estrus have yet not been reported in sheep. Different species of female livestock has her own estrous cycle and proper mode for estrus. Hence, the objective of the present study was to compare the efficacy of three estrus synchronization protocols, CIDR9+PGF_2α+eCG; CIDR14+ eCG and GnRH+PGF_2α, in Hu sheep.

Eighty fertile and multiparous Hu sheep, ageing 2.5±0.30 years, with a mean body condition score of 3 (scale ranging from 1=emaciated to 5=obese) described by Russel et al., (1969), were conducted in this study during November 2017 through May 2018. Ewes were placed in yards under a roof in a ventilated barn at the commercial farm of Jiangsu Qianbao animal husbandry Co., LTD located in Yancheng of Jiangsu Province of China. During the period of the experiment, the diets of animals was based on chopped green maize, bean pulp, rapeseed meal and bran as roughages and concentrate supplements at a daily level of 500 g/ewes (15.05% crude protein) to meet their daily energy and protein requirements and had free access to clean water and mineral salt. The experiments on the administration and handling of sheep were assessed and approved by the Animal Ethics Committee of Experimental Animal of Yangzhou University (Yangzhou, China; Code No. AW 20171015).
 
Synchronization protocols
 
According to the protocols of estrous synchronization, ewes were randomly allocated into four groups (n=20 per group). Briefly, CIDR9+PGF_2α+eCG-group ewes were received short-treated CIDR constructed with silicone elastomer impregnated with 0.4 g natural P4 for 9 days period combination with intramuscular first injection of 30 μg dose of PGF_2α on 8 day followed with the second injection of 350 IU dose of eCG at CIDR withdrawal time on 9 day. CIDR14+eCG-group ewes were treated with long-treated CIDR for 14 days and injection of 350 IU dose of eCG at CIDR withdrawal. GnRH+PGF_2α group ewes were conducted by intramuscular administration of 6-μg GnRH and injection of 30 μg PGF_2α on 8 day. The fourth group was control group received no further treatment. CIDR or GnRH treatment applied in treated-group was defined as day 0, the experimental design is shown in Fig 1. Before experiment, normal estrus cycle of the ewes had been confirmed twice and they had not been fertilized in order to ensure the animals employed in our experiment didn’t have ovarian disease.
 

 
Detection of estrus and artificial insemination
 
Estrus activity was detected using teaser rams (one ram per approximately 15 ewes) equipped with harnesses beginning with 12 h after CIDR removal or injection of exogenous hormone and checked every 12 h, twice a day until the end of the signs of estrus. Expressing a sign of standing estrus ewes was recorded.
   0-24 h, 24-48 h, 48-72 h, respectively) in each treatment group was recorded.
       
Three proven healthy and fertile rams were chosen for ejaculated sperm collection by artificial vagina. Obtained semen was immediately evaluated macroscopically and microscopically. A total of 6 fields of view were quantified per chamber by electron microscope (OLYMPUS, TOKYO 163-0914, JAPAN). Approximately 500 sperms were analyzed and done in duplicate. The motility of sperm (forward progressive sperm / the total analyzed sperm) is up to 0.8, the smell is slightly fishy and the color and luster looks like milk white, which could be used for artificial insemination (AI). The semen was diluted for 200×106 live normal motile sperm per 0.1 mL with commercial extender (Eigelbfreies Verdunnerkonzentratfur Bullensamen androMed, Minitube, Germany). Diluted semen was stored at 4^oC and used within 3 days. At the time of AI, Sperm motility was reanalyzed and confirmed to be up to 0.8. Ewes were submitted to laparoscopic AI with 0.3 mL diluted semen.
 
Pregnancy diagnosis
 
Pregnancy diagnosis was carried out on day 28 after insemination and confirmed on day 60 through transrectal B-mode real ultrasonography using a dual-frequency (3.5/5.0 MHz) convex abdominal probe (NL219M/DFLM36 Tinga Vet 50 s, Yum medical in Italy). The purpose of such regular detections performed was to identify the number of fetuses and to confirm the amount of lambing.
   
Statistical analysis
 
Estrus detection rate, pregnancy rate, conception rate, twining rate, lambing rate, fecundity and prolificacy were analyzed by Chi-square. The other statistics were evaluated by ANOVA as repeated-measures with the Tukey HSD multiple comparison test in all measured parameters, using SPSS 22.0. A probability of P<0.05 was regarded as significant difference. Date represented in duration of pregnancy and hormones levels except percentages are expressed as the mean±standard error.

Detection of reproductive performance
 
Ewes failed to keep CIDR devices in the vagina were eliminated from our experiment (n=1 both in CIDR-based protocol groups). Statistics shown in Table 1 illustrate that the estrus rate (84.21%, 89.47%, 90%, respectively), pregnancy rate (47.37%, 63.16%, 65.00%, respectively), conception rate (56.25%, 70.59%, 72.22%, respectively), lambing rate (56.25%, 70.59%, 72.22%, respectively) of three treatment groups have no significant difference compared with control (75.00%, 60.00%, 80.00%, 80.00%, respectively). It is noted that even though the estrus rate between CIDR14+eCG (89.47%) and GnRH+PGF_2α (90.00%) were almost equal, the implement of intravaginal devices may trigger several problems, including the production of chemical residues and vaginitis that may threaten to female health, which encourage the usage of PGF_2α as an alternative, clean and safe for estrus synchronization.
 

       
Most notably, CIDR+PGF_2α+eCG and CIDR+eCG (145.78±1.56, 144.42±0.67, respectively) have significantly shorter (P<0.05) duration of pregnancy than control (147.20±1.52), but GnRH+PGF_2α (146.31±1.55) has no effect on duration of pregnancy compared with control. Furthermore, CIDR+eCG (144.42±0.67) has shorter (P<0.05) duration of pregnancy than the remaining treatment groups. This may due to the serum levels of P4 increased in CIDR-treated (14 days) ewes at 0 hour (Swelum et al., 2015); The increasing P4 level during early pregnancy, which can decreases the lost of embryo and increases pregnancy rate and fertility (Ataman et al., 2013). Therefore, during the later of pregnancy, there will not enough nutrition and space for the lamb to growth, which leading to shorter duration of pregnancy. With respect to fecundity, CIDR+eCG and GnRH+PGF_2α did not dramatically change the fecundity of ewes, while CIDR+PGF_2α+eCG significantly decreased (P<0.05) the fecundity of that compared with the control.
 
Detection of estrus of various times
 
Effect of three treatments on percentage of ewes behaving estrus responses within various time of estrus detection post-treatment, 0-24 h, 24-48 h, 48-72 h, was descripted in Fig 2. It suggested that estrus responses in CIDR9+PGF_2α​+eCG, CIDR14+eCG and GnRH+PGF_2α​ groups rose gradually to attain their significantly higher percentages (56.3%, 52.9% and 66.7%, respectively) during 24-48 h post-estrous detection, afterwards, they fell down. Besides, GnRH+PGF2a would cause the estrus cycle more synchronized and the estrus time is focused at the 24-48 hours after PGF_2α treatment with the estrus rate up to 66.7%. This in line with the results, reported by Evans (1988), Menchaca and Rubianes (2004), Zeleke et al., (2005), that both the use of equine chorionic gonadotropin (eCG) and gonadotropin releasing hormone (GnRH) treatments could provide a more compact ovulation in ewes, thereby providing the potential to increase the pregnancy rates following AI in sheep. From the investigations on sheep folliculogenesis, it has been known that a follicle is in its active growing phase for the following four days to reach its maximum diameter at the fifth day after the insertion of a CIDR (Martinez-Ros​ et al.,  2018). It should be noted that the differences in size between dominant and subordinate follicles are very small and periods of effective dominance are very short (Gonzalez-Bulnes et al.,  2004). Furthermore, when sheep treated by GnRH+PGF_2α, the luteal tissue that forms as a result of the GnRH administration is responsive to PGF2_α​ and is capable of undergoing luteolysis (Husein and Kridli, 2003). However, the response to GnRH is dependent upon the stage of the cycle at which it is administered. There have been reported that about half of the ewes had active corpora lutea at the time of GnRH administration (Alminer et al., 2005; Titi et al., 2010). Accordingly, GnRH+PGF_2α-group appear to delay the phenomenon of ovulation, onset of oestrus.
 

Although CIDR-based short-term (9 days), CIDR-based long-term (14 days) and GnRH+PGF_2α estrus syn chronization protocols are all efficient in Hu sheep, taking the discussion above into account, GnRH+PGF_2α based protocol would result in the estrus cycle more compact synchronization in Hu sheep and the estrus time is concentrated at the 24-48 hours after PGF_2α treatment with the estrus rate up to 66.7%.

The authors would express their appreciation to the Jiangsu Qianbao animal husbandry Co., LTD for their assistance in animal care. The research was also supported by funding from Jiangsu Agricultural Industry Technology System [JATS (2021) 491] and the Priority Academic Programme Development of Jiangsu Higher Education Institutions (PAPD).

None.