**7.1 Cattle as a model animal for multiple ovulation**

As a uniparous species, cattle produce only one progeny in most cases, resulting from ovulation of a single follicle during the pregnancy. Nevertheless, the natural incidence of twin or triplet calving in cattle is mainly due to multiple follicular ovulations concerning breed differences, age of dam, parity, season, the effects of feeding and management systems, geographic location of raised animals, and other environmental effects [62]. Specifically, the incidence of double birth was observed as approximately 1% in beef cattle [92]. In comparison, this rate was determined as 4–5% on average, ranging from about 1% for heifers to nearly 10% for older cows in dairy cattle [93, 94]. Several studies were conducted concerning underlying causes of multiple ovulation rates, particularly twinning rates in cattle by selecting

genetically highly polymorphic animals [63], using trans-rectal ultrasonography or quantifying by circulating AMH concentrations, utilizing embryo transfer techniques [65], or utilizing hormonal treatments [64].

The ovulation rate is closely related to the twinning rate in cattle due to the high genetic correlation between ovulation and twinning rates, ranging from 0.75 to 1.0 [95]. Although the genetic control of multiple ovulation in cattle by major genes has long been the subject of research, and there has been significant interest in the mechanism underlying multiple ovulation in bovine species [70], genes with significant effects on ovulation rate have unfortunately not been identified until recently [96].

### **7.2 QTL studies about twinning and ovulation rates**

The selection of genetically superior animals in terms of twinning frequency has been practiced in long-term experimental herds in different countries. For this purpose, various research herds for multiple ovulation studies have been implemented to be established in various countries for four decades. These herds were begun to set up in the early eighties to select for increased twinning rates in France [97], Australia [76], New Zealand [98], and Meat Animal Research Center (MARC) of the USDA-ARS in the USA [99] to develop effective genetic strategies to improve production efficiency including twinning and ovulation rates, meat quality, and animal health in dairy and beef cattle production. MARC twinning population initiated with a total of 307 well-suited cows from twelve different experimental beef, dairy, and dual purposes breeds to study involved in follicular development and recruitments and identify genes affecting primarily twinning rate; later taken into account of ovulation rate in 1981 [63, 100]. These cows were selected based on their high twinning frequencies. The twinning rate can be defined as sequential events due to ovulation, conception, and embryonic survival [101]. Sires whose dams were founders of the herd and sires whose daughters had high twinning rates were used for breeding the founder cows. In addition, semen collected from sires that mainly originated from Swedish and Norwegian breeds was used in the project. The founder breeds in the herd were mainly Holstein (18%), Swedish Red and White and Norwegian Red (12.8%), Swedish Friesian (16.1%), Pinzgaurer (18.4%), Simmental (15.8%), Charolais (5.3%), Angus and Hereford (8.3%), and other breed crosses (5.3%) [102]. The primary objective of the research was to increase the twinning rate in the herd. Therefore, they selected animals based on twinning performance. However, later on, they also evaluated animals' ovulation rate records for 8 to 10 estrous cycles since ovulation rate is highly genetically correlated with the twinning rate (0.90) [58]. Thus, they used an animal model with multi-trait repeated records to predict breeding values for twinning rates in 1990. By applying this methodology, they were able to use information not only from the individual but also from all available relatives for twinning and ovulation rates. The most significant advantage of using ovulation rate records as an estimator of twinning rate is to reduce generation interval and reduce the number of cows retained for several generations. The estimated twinning rate was about 4% in 1984. But this prediction rose linearly to 35% in 1996 [100]. In the latest report, all the cows with lower estimated breeding values (EBV) were culled from the herd. Thus herd size was reduced from 750 to 250 cows giving birth annually. The twinning rate then was enhanced from 35% to over 50% annually since 1997 [103, 104].

Many studies have been conducted to identify ovulation rate and twinning rate QTL in different cattle populations. Several genomic regions for putative ovulation rate were detected on BTA7 and 23 [105], on BTA5, 7, and 19 [106], on BTA5 [107, 108], on BTA7, 10, and 19 [109], on BTA14 [101] for ovulation rate using the USDA

*The Incidence of Ovulation and Detection of Genes Associated with Ovulation and Twinning… DOI: http://dx.doi.org/10.5772/intechopen.99936*

Meat Animal Research Center (MARC) twinning herd, a herd with a substantial contribution from Holstein–Friesian and Norwegian Red breeds [110]. A suggestive twinning rate QTL on BTA5, 7, 12, and 23 have been identified in the Norwegian dairy cattle population [111, 112]. Twinning rate QTL based on genome-wide searches have also been observed on BTA5, 7, 19, and 23 [113], on BTA8, 10, 14, 21, and 29 [114], on BTA2, 5, and 14 [115] in North American commercial dairy cattle populations; on BTA6, 7, and 23 in the Israeli-Holstein cattle using daughter design [116], on BTA20 and 28 in the INRA experimental herd selected for twinning [117]. In studies using composite MARC herds, it was determined that cows producing twins based on genetic selection for high twinning and ovulation rates over multiple generations produced about two-fold more secondary follicles than animals in the control groups. The probable reason for the higher twinning and ovulation rates in this herd may be the combined effects of multiple genes associated with these quantitative traits [63, 100]. Multiple positional candidate gene regions associated with ovulation rate, twinning rate, and multiple birth rates in various cattle breeds have been identified by linkage analysis, interval mapping, linkage disequilibrium (LD) analysis, the combined linkage-linkage disequilibrium analysis (LDLA), and GWAS analyses even if only a few have been replicated. Depending on the statistically significant level, the QTL or single nucleotide polymorphisms (SNPs) determined in the studies so far were diverse throughout the bovine genome. They spanned about 18 of the 30 bovine chromosomes given in **Table 1** [89].

It is noteworthy that a crucial QTL region was detected on BTA5 in the MARC experimental herd, commercial dairy cattle populations raised in North American and Norway. Some of the founder sires in the composite MARC population were originated from Scandinavian countries whose progenies gave multiple births. Therefore, the probability of detecting the same QTLs in future studies is quite high due to sharing a significant portion of the founder genes in two different populations [63]. Furthermore, different studies have reported that IGF-1 as a candidate gene (especially the 2nd intronic region) in BTA5 is substantially associated with the twinning rate in US Holstein cattle [118, 119]. The presence of several QTLs for twinning rate and ovulation rate was detected, which were spanned 24 out of the 30 bovine chromosomes as a result of studies using high-throughput single nucleotide polymorphism (SNP) genotyping throughout the genome based on linkage (LE) and linkage disequilibrium (LD) analyses.

### **7.3 Novel candidate genes affecting multiple births in cattle**

In cattle, twin or triplet births are naturally occurring reproductive processes, although not a joint physiological event in bovine. Models derived from the study of high prolific sheep breeds provide a framework for searching the regulation of follicular development in monotocous species, such as in cattle or humans.

A highly fertile cow named 'Treble' was born in 1993 at one of the cattle herds in New Zealand. Although the breed's origin is unknown, it has been assumed to likely include a hybrid of Hereford, Holstein, Angus, and Jersey breeds based on the coat color pattern. Treble calved three sets of triplets her life span as one heifer and two stillborn calves at the first time in 1995, two heifers and one bull, named as Trio at the second time in 1996, all stillborn calves due to considerable difficulty during the delivery period at the third time in 1999. Treble was cloned later, and two clone progenies were born in AgResearch Centre, NZ, in 2000. On the other hand, a son of a highly prolific cow named the 'Triple' was bred with a group of cows with high calving rates that had several progenies by 2008. Thirteen daughters out of his total of forty-four daughters produced a total of fifteen twin and six triplet sets, where triplet calving were 29% of all multiple calving, supporting the idea of a naturally-occurring


### **Table 1.**

*Chromosomal locations of quantitative trait loci (QTL) and single nucleotide polymorphisms (SNP) associated with ovulation rate, twinning rate, and multiple birth rate in various cattle breeds [89].*

major bovine allele contributing to a high fecundity rate in a family of cattle with triplet calving ability throughout the generations in New Zealand. The possible scenario for this situation might be that a gene or set of genes should be segregated as a single copy from a dam (Treble) to some descendants through its son (Trio) for single gene inheritance. Moreover, such a unique gene allele is expected to be segregated as dominant or partially dominant in female animals [70].

### *The Incidence of Ovulation and Detection of Genes Associated with Ovulation and Twinning… DOI: http://dx.doi.org/10.5772/intechopen.99936*

Several daughters (131) of Trio were born by AI in the USA by following the importation of his sperms at a University of Wisconsin (UW)-Madison research farm from 2008 to 2011. The research reports that a significant bovine allele for high ovulation was identified and mapped on a 2-Mb window on BTA10 (+1.02 CL per cycle for carriers vs. noncarriers for the marker allele of the high ovulation rate) by using fine mapping techniques employed the animals raised at UW-Madison research farm [120]. Thus, the daughters of Trio proved that there was evidence of a high-fecundity allele transmitting on BTA10 that had a major influence on multiple ovulations in cattle [96]. The detected location was not overlapped with any major genes previously reported for the high ovulation rate and litter size in prolific sheep breeds. Eventually, in addition to the noteworthy reproductive performance of Treble, all of her descendants, including Trio, also displayed extraordinary reproductive performance. Therefore, the members of the Treble family with highly reproductive ability should be heavily employed in gene mapping studies to discover major genes with high fecundity rates. It can provide a significant resource for the subsequent investigation of genetic diversity in bovine productivity [70]. In the follow-up study, the location of a major gene for high ovulation rate was strongly detected at 1.2 Mb region of BTA10 using halfsib daughters sired by a bull that assumed to be carriers of the Trio allele due to a single mutation. It is noteworthy that the novel region obtained does not overlap with any major gene previously reported, which significantly affecting ovulation rates in ruminants. Thus, the study reports that the newly identified regions could be employed to track inheritance patterns for multiple ovulation rates using from the carrier father's lineage since the screening of the aforementioned candidate gene consist of any functionally putative causative mutations in the coding region and 5′ or 3′ flanking regions, reminding that the polymorphic SNP region might affect the expression level of any candidate gene controlling the high reproductive performance of animals [96]. When the follicular and hormonal dynamics of animals carrying the high prolific Trio alleles were examined in animals raised at UW, the Trio carrier animals displayed multiple ovulation. The carriers produced more dominant and ovulating follicles with smaller diameters and volumes in this process due to the slower follicle growth rate close to the beginning of deviation during the entire follicular wave. In the study, even if the deviation times were similar between heterozygous bearing allele from Trio and half-sibling noncarriers, a significant increase in the selected number of multiple dominant ovulatory follicles in cow having Trio allele was reported to be associated with the enhanced concentration of FSH secretion close to the deviation time in the follicle. There was also evidence that smaller-sized follicles had more LH receptors in animals carrying the Trio allele than noncarriers, supporting the potential novel physiological mechanisms causing the production of multiple ovulatory follicles in the Trio allele carriers [89].

This newly identified candidate region covering 1.2 Mb in BTA10 contains seven protein-coding genes, of which three of them might be taken into account as putative candidate genes. These genes are the small-mothers against decapentaplegic (SMAD) family member 3 (SMAD3), SMAD family member 6 (SMAD6), those of which are the primary signal transducers for the receptors of the transforming growth factor-β (TGFβ)/Bone Morphogenic Protein (BMP) superfamily ligands [123], and IQ motif containing H (IQCH), which is strongly related with the first menstrual cycle in human females [124]. The other follow study stated wellconserved SMAD6 gene, which plays a crucial role in preventing the BMP/SMADdependent signaling pathway, was 9.3 times more expressed in carrier animals for the high fecundity Trio allele versus noncarriers using animals in UW-Madison research farm by applying quantitative real-time PCR technique.

Ultimately, the effect of over-expression of the SMAD6 gene displayed a similar impact of causative mutations on the functions of BMP15, BMPR1B, and GDF9 genes as part of a signaling pathway that may alter the incidence of ovulation rate upward in prolific sheep breeds [125].

In another study to determine the genetic basis of the observed increases in twinning and calf mortality in Italian indigenous Maremmana cattle breed, the most significant SNP markers (Hapmap22923-BTA-129564) were located near two genes, ARHGAP8 and TMEM200C on BTA24, which could be putative functional candidates for cattle twinning rates [121]. Furthermore, in a very recent study, the researchers detected a major QTL mapped to a 70 kb window between 31.00 and 31.07 Mb on BTA11 for multiple maternal births, explaining approximately 16% of the total genetic variation based upon linkage-disequilibrium analysis (LD) using the whole-genome sequence information of the Swiss cattle population. The identified QTL includes the LHCGR and FSHR genes as functional candidate genes. Precisely, a regulatory variant in the 5′ non-coding region of LHCGR is predicted as a potential causative mutation for the QTL region [122].
