Remarkable seasonal variations are observed in the reproductive activities of small ruminants living in temperate latitudes (Luridiana et al. 2015). Thus, a great alteration is induced by lamb reproductive seasonality and its consequent meat market price. Alternatively, out-of-season reproduction is induced through a wide use of hormonal treatments in some countries. However, there is a need for a search for some other methods due to the increasing demand for free-hormone products (Martinez-Royo et al. 2012). Nonetheless, identification of informative genetic markers has been suggested to improve selection efficiency for reducing breeding seasonality in sheep (Moradi et al. 2014). In a response to photoperiodism, animals are engaged in the secretion of differential melatonin to regulate their reproductive activities (Saxena et al. 2014). The light signals relayed to the pineal gland by several neurons from the retina, known as photoperiodic information, are translated into the daily cycle of melatonin secretion, the level of which is high at night and low during day time (Shahroudi et al. 2006). Accordingly, the secretion of gonadotrophin-releasing hormone (GnRH) is controlled by the seasonal rhythms of melatonin secretion that serves as a seasonal messenger in endocrine tissues at the hypothalamic level (Luridiana et al. 2014). The alternating presence or absence of female ovulation and male sperm production is dependent on luteinizing hormone secretion, the levels of which are induced by the corresponding changes in GnRH release (Shahroudi et al. 2006). Melatonin reproductive effects occur in the premammillary hypothalamus (Luridiana et al. 2015) and the hypothalamic suprachiasmatic nucleus is the site of the circadian clock where melatonin circadian effects are mediated by the pharmacologically specific G-protein-coupled melatonin receptors with high affinity (Jia et al. 2012), through which melatonin exerts its effects when binding to them. However, it seems that the reproductive activity is only regulated by MT1 from among the receptors involved (Luridiana et al. 2015). In an investigation for sheep sexual activity, Notter et al. (2003) showed that the genotype of this gene might become as a particular marker based on the association between different allelic forms at MT1 locus and sheep reproductive activity. Reppert et al. (1994) were the first to clone melatonin receptor 1A gene (MTNR1A) as a high-affinity subtype in mammals. It should be noted that from among any other melatonin receptor subtypes like MTNR1B and MTNR1C, only MTNR1A appears to be engaged in the seasonal reproductive activity regulation (Saxena et al. 2014; Trecherel et al. 2010). An association between MTNR1A gene and seasonal reproductive activity has been evidenced by several studies on different animal species (Luridiana et al. 2014). Circadian variations in melatonin concentrations have been repeatedly proposed to be caused by MTNR1A as a candidate gene via photoperiodic control of seasonality (Mura et al. 2014). MTNR1A gene, consisting of two exons divided by a large intron, has been mapped in the ovine chromosome 26. The exon one shows low degree of polymorphism. It has been shown that exon 2 of MTNR1A gene, which is involved in the coding process of ovine MT1 receptors is highly polymorphic, while changes in the second exon would make the differences in the structures of the receptors. The 2 restriction fragment length polymorphism (RFLP) sites for MnlI and RsaI enzymes are derived from exon 2 of the gene encoding MT1 receptor in sheep. The characteristic pattern of digestion by this enzyme is characterized by a mutation at MTNR1A/MnlI site, which leads to the absence (–or M) of MnlI specific cleavage site at position 605 of the coding sequence (Hatami et al. 2014). The 3 genotypes of “M/M”, “M/m” and “m/m”, which were previously described as +/+, +/− and −/− (Trecherel et al. 2010), are discriminated by the presence or absence of this mutation. Sheep, goat and buffalo were selected to carry out the studies of relationship between this gene polymorphism and reproductive activity. In this regard, a reproductive association with the polymorphic sites identified in MTNR1A gene was shown by some sheep breeds like Merinos d'Arles, small tailed Han sheep, Awasi and Sarda (Saxena et al. 2014). A local sheep breed is Shal, which constitutes a population of more than 600000 heads in Iran. The breed is large-sized, fat-tailed, and predominantly black or brown with white spots on the front head, while being well-adapted to harsh climates. It is mainly raised for its meat as the most important protein source in Iran. Following a random exposure of Ewes, which usually lamb 3 times every 2 years, to 18-month rams, lambing occurred within one season, i.e. from mid-January to mid-March (Hossein-Zadeh, 2015). One of the breeds renowned for its long breeding season, early sexual maturity and high prolificacy is Romanov. However, poor carcass quality and relatively low growth rate are of the typical features of this breed in comparison to the traditional meat species. Application of a commercial crossing approach with the meat-type breeds is a simplest and fastest way of improving growth and carcass quality in Romanov lambs (Kuchtík et al. 2012). Crossbreeding between Romanov and Shal were used to improvement of prolificacy in Shal breed. In the current study, we considered the polymorphism of exon 2 of MTNR1A at MnlI site as a marker for prolificacy and investigated its relationship with the production traits of Shal and Romanov crossbreeding.
MATERIALS AND METHODS
Experimental animals and their management:
This experiment was carried out at a sheep farm in Ismaeel Abad (longitude: 49.66˚ E and latitude: 36.46˚ N) located in Qazvin province, Iran. Shal and Romanov crossbreeding were done on 600 Shal ewes. To this purpose, Shal ewes (1-2 years old) were laparoscopically and artificially inseminated with the semen of Romanov breed. An integration of natural pasture and concentrate feed under natural photoperiod was followed the ewes since birth. All the lambs were born indoors and a similar nutrition status was considered for all sampled individuals within each breed.
Preparation, amplification, and digestion of genomic DNA
A total of 90 ewes, either Shal (n=50) or Shal × Romanov (n=40), were used. Five mL of blood was collected from the jugular vein in ethylenediaminetetraacetic acid (EDTA)-coated tubes. Ovine genomic DNA was extracted using phenol-chloroform method as previously described by Psifidi et al. (2015) and maintained at -20 ˚C until use. Approximately, 200 ng of genomic DNA was subjected to polymerase chain reaction (PCR) by using specific primers synthesized by Sina colon, Iran. The primers werethose used by Messer et al. (1997) (sense primer 5´-TGTGTTTGTGGTGAGCCTGG-3´ and antisense primer: 5´-ATGGAGAGGGTTTGCGTTTA-3´) from the sequence of Exon II of the ovine MTNR1A gene (GeneBank U14109). Polymerase chain reaction (PCR) was carried out in a volume of 25 μl containing 200 ng of DNA, 1XPCR buffer, 2.0 mmol of MgCl2, 0.2 mmol of each dNTP, 10 pmol of each primer, and 2 U of TaqDNA polymerase (Fermentas, Canada). The PCR conditions were as follows: initial denaturation at 94 ˚C for 5 min followed by 35 repeated cycles of denaturation at 94 ˚C for 1 min, annealing at 62 ˚C for 1 min, extension at 72 ˚C for 1 min, and final extension at 72 ˚C for 10 min on a thermal cycler (Bio-Rad, T100). The PCR products were resolved by electrophoresis on 2% agarose gel in parallel with 250 bp of DNA marker ladder (Fermentas). Then, they were separated by electrophoresis on 2% agarose gel. After amplification, 7 µL of the PCR product was digested with 2 units of MnlI endonuclease at 37 ˚C for 4 hours following a deactivation process at 65 ˚C for 20 minutes.
For the genotyping of the study samples, the digested fragments were electrophoresed on 3% agarose gel and stained with ethidium bromide. Allelic frequencies were determined by direct counting of the observed genotypes. The X2 test was used to determine Hardy-Weinberg (HW) equilibrium of the mutation by using following equation (GenAllex Software).
The signiﬁcance of differences between the treatments was evaluated using the t-test. The results were expressed as mean ± SEM and the statistical signiﬁcance was accepted at P < 0.05. The data were analyzed using a statistical software (SPSS, 2011).
RESULTS AND DISCUSSION
PCR-RFLP analysis of exon 2 of MTNR1A gene
In current study 824 bp fragment of exon 2 of Shal and Shal × Romanov MTNR1A gene were analyzed by RFLP (Figure 1). The presence and absence of 303 bp fragment were referred to as alleles m and M, resulting in 236 and 67 bp products. Thus, the 3 genotypes of MM, Mm, and mm were detected based on 236, 236 and 303, and 303 fragments, respectively. In our study, the 2 genotypes of MM and Mm were detected in the study population of Shal with the frequencies of 0.78 and 0.22, respectively (Figure 2 and Table 1). However, MM and mm genotypes were detected in the crossbreeds under study with 0.125 and 0.0875 frequencies, respectively. Mm and mm genotypes were not detected in Shal × Romanov and Shal breeds, respectively (Figure 3 and Table 1). MM and Mm frequencies were significantly higher in Shal compared to Shal × Romanov crossbreeds, and that of mm was significantly higher in the study crossbreeds (P<0.05). The allelic frequencies of 0.89 and 0.125 were observed for M allele and 0.875 and 0.11 for m allele at MTNR1A locus in Shal and Shal × Romanov crossbreeds, respectively (Table 1). Contrary to our results, Chu et al. (2006) demonstrated an association between MM genotype and non-seasonal estrus in ewes, as well as a relationship between mm genotype and seasonal estrus in ewes when investigating non-seasonal estrous breeds (Small Tail Han and Hu ewes) and seasonal estrous breeds (Dorset, Suffolk, and German Mutton Merino ewes). Many studies indicated an association between the homozygous genotype for the absence of a polymorphic MnlI site at position 605 of exon 2 of MTNR1A gene and seasonal anovulation in ewes (Chu et al. 2006). In our research, the X2 test confirmed that only the study Shal sheep population was in HW equilibrium. Perhaps, this was due to the limited number of crossbred population and non-random mating. Nevertheless, congruent with our results, Carcangiu et al. (2009) reported that the ewes of MM genotypes lambed in autumn following an oestrus in spring. Having a very high frequency (0.81%) among their study ewes, M allele surely played a very important role in this process. Though Notter et al. (2003) demonstrated that the various sheep breeds reared in North America were sufficiently influenced by a single M allele leading to a reproductive seasonality, Pelletier et al. (2000) found that MM homozygous ewes of Merino d’Arles sheep breed showed oestrus in spring. In their study on native Iranian breeds of sheep, Ghiasi et al. (2006) demonstrated that the sheep, which genetically had a potential to show estrus during short and long days, indicated 2 genotypes at MTNR1A locus with the frequencies of 0.7 (MM) and 0.3 (Mm) in the Karakul breed, and 0.58 (MM) and 0.42 (Mm) in the Shal breed. Also, in another study on native Iranian breeds, i.e. Zel and Naeini, Moradi et al. (2014) reported that M allele was predominant in either Zel or Naeini lambs.
Figure 1 Electrophoresis pattern of PCR product of exon 2 of MTNR1A gene (824 bp) in Shal and Shal × Romanov sheep (2% agarose gel) Lanes 1-3 and 4-5, PCR amplification product for Shal and Shal × Romanov, respectively, and M: DNA molecular marker (250 bp)
Figure 2 RFLP Genotyping of MTNR1A gene by MnlI in Shal breed
Table 1 Allele and genotype frequencies of exon 2 of MTNR1A gene for MnlI site in Shal and Shal × Romanov breeds
The genotypic frequencies for MM, Mm, and mm genotypes were 0.52, 0.25 and 0.23 in Zel breed and 0.60, 0.22 and 0.18 in Naeini breed. Furthermore, they showed that MM genotype had the lowest mean for litter size significantly. In general, the highest mean measurements for litter size were obtained from mm followed by Mm, whereas the minimum mean measurements were achieved from MM in both assessed breeds. Even in Sarda sheep breed exhibiting an anoestrous period in late-winter/spring, polymorphisms within MTNR1A gene lead to some advances in reproductive activity resumption (Carcangiu et al. 2009). Additionally, Luridiana et al. (2015) evidenced an relationship between MTNR1A gene polymorphism and a resumption of reproductive activity in adult Sarda sheep breed in spring.
Figure 3 RFLP Genotyping of MTNR1A gene by MnlI in Shal × Romanov breed
However, no association between MNTR1A polymorphisms and seasonality of reproduction in Ile-de-France ewes was found by Hernandez et al. (2005). Moreover, some European breeds have not proven any impacts of MTNR1A gene polymorphisms on their reproductive activities. The difference might be well attributed to breed traits or body and environmental conditions. Thus, there is a need to expand the knowledge of the effects of MTNR1A gene polymorphism for the clarification of the mechanisms regulating the different reproductive responses of the different sheep breeds (Mura et al. 2014).
Association between MTNR1A gene polymorphism and birth and body weights
There are restricted opportunities for a selection within the breeds due to the fact that less heritability (nearly 0.05-0.15) occurs for most reproductive traits compared to many other traits. Nonetheless, to optimize reproductive potentials, a within-breed selection must be done after crossing the divergent breeds to the genetic potentials rapidly reset for those traits, through which basic changes in litter size or seasonal breeding patterns are best achieved. Another opportunity can be gained by quickly adjusting genetic potentials via various mutations that affect the ovulation rate and litter size of sheep though a careful breeding management is required, particularly for production traits (Notter, 2012). Our results revealed that MnlI marker site in Shal × Romanov breeds had a significant effect (P>0.05) on a 3-month body weight (BW3) as ewes with MM genotypes had significantly higher BW3 than mm individuals. However, no significant differences were observed between MM and mm marker sites for 6-month birth and body weights (BW6) (Table 2). The same results were observed for Shal breed as MM and Mm genotypes were detected for BW, BW3, and BW6 traits (Table 3).
Table 2 Production performance (Mean±SEM) of Shal × Romanov breed
BW: birth weight; BW3 and 6: body weight at 3 and 6 months of age.
SEM: standard error of the means.
NS: non significant.
Table 3 Production performance (Mean±SEM) of Shal breed
BW: birth weight; BW3 and 6: body weight at 3 and 6 months of age.
SEM: standard error of the means.
NS: non significant.
Although in our study, BW3 was affected by MnlI genotypes in both Shal and Shal × Romanov breeds, the production traits of Shal and Shal × Romanov breeds were not affected by MnlI genotypes based on BW and BW6 traits. Also, they were not influenced by mm or Mm genotypes in the current studies. In their study on the seasonal reproduction of Zandi breed, Hatami et al. (2014) reported that MTNR1A/MnlI marker site had a significant effect on BW1 as the ewes with Mm genotypes were found to have higher BW1 compared to MM individuals. No significant association was observed between MTNR1A/MnlI marker siteand the other study traits, including BW, BW3, and BW6.
In the current research, the 2 genotypes of MM and Mm were detected in Shal population involved in seasonal reproduction. However, MM and mm genotypes were detected in Shal with Romanov crossbreeding. Thus, mm genotype observed as a measurement for litter size in sheep breeds successfully introduced into the crossbreeds. Therefore, based on BW and BW6 traits, the production traits of Shal and Shal × Romanov breeds were not affected by MnlI genotypes. Nevertheless, further studies are recommended to reveal the relationship between MTNR1A gene polymorphism at MnlI site and reproductive performance of Shal × Romanov ewes.
We would like to thank Amir Hossein Ahadi for his helpful comments during the research. This project supported by Iran national science foundation.