Summary and Implications

Two major components of litter size - ovulation rate and embryonic survival - have been used in a selection index project ongoing for several generations at the University of Nebraska-Lincoln (UNL). Additionally, the Chinese Meishan breed is one of the most prolific breeds, producing four to five more pigs per litter than white crossbred females.

We investigated the role of the gonadotropin-releasing hormone (GnRH) receptor and gonadotropin subunit genes in determination of ovulation rate between lines of swine. Ten UNL Index and Control line white crossbred gilts and 12 Meishan gilts were ovariectomized following three (Index and Control) or 6 (Meishan) successive estrous cycles. After a 21-day recovery period, gilts from each line were treated with either a specific GnRH antagonist (SB-75; 10 \ig/kg of body weight) or 0.9% saline at 60, 36 and 12 hours prior to slaughter. Blood samples were collected prior to the first treatment and at slaughter before anterior pituitary collection. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were determined by radioimmu-noassay and RNA was extracted from anterior pituitary tissue. In all lines, LH was reduced to basal levels by SB-75 treatment, confirming the efficacy of SB-75. In contrast, levels of FSH decreased only in Control gilts following treatment with SB-75.

Pituitary levels of GnRH receptor and gonadotropin subunit gene expression were measured by quantitative PCR. Levels of gene expression for the GnRH receptor and gonadotropin subunits decreased following treatment with the GnRH antagonist in pituitaries of gilts from the Index and Control lines; however, these values remained unchanged in pituitaries from Meishan gilts. Identification of unique genetic changes in swine strains with increased ovulation rates, such as the Chinese Meishan and the UNL Index selection line, may allow for a better understanding of prolificacy. This critical information may also be used to enhance litter size in other lines of pigs and improve efficiency of pig production.

Background and Introduction

Prolificacy is an important economic measure of productivity in the pork industry. However, many generations of selection are required to increase the number of live born piglets per litter within an applied breeding program. Thus, it is important to identify the genes and underlying biological mechanisms contributing to increased litter size. While many factors can influence prolificacy, a primary component of litter size is ovulation rate, or the number of oocytes (eggs) available to be fertilized after insemination. From a research perspective, ovulation rate can be measured via visualization of ovaries by surgical procedures (i.e., laparotomy or laparoscopy) or ultrasound. In addition, this trait can be improved in a number of different ways, including nutrition (flushing), hormonal treatment (superovulation), and genetic selection. Despite its importance as a primary component of litter size, however, there is very little known about the genes influencing increased ovulation rate.

Ovulation rate is influenced by circulating levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH), also known as the gonadotropins. The production of these hormones is controlled by the reproductive axis (Figure 1), consisting of the hypo-thalamus, anterior pituitary gland, and gonads (ovaries or testes). Specifically, gonadotropin-releasing hormone (GnRH), released from the hypothalamus, binds to its receptor on gonadotrope cells of the anterior pituitary gland. Upon binding to its receptor, GnRH stimulates the expression of the GnRH receptor gene itself, as well as the subunit genes that lead to the production of FSH (common alpha- and FSHbeta-subunits) and LH (common alpha- and LHbeta-subunits). The secreted gonadotropins then act on the ovaries to recruit follicles (FSH) or induce ovulation (LH). Steroids, produced by the ovaries, such as estrogen and progesterone provide feedback at the level of both the hypothalamus and anterior pituitary gland to regulate subsequent production of GnRH and gonadotropins, respectively. Therefore, reproductive function is highly dependent on the interaction of GnRH and its receptor.

Sensitivity, or number of GnRH receptors, present on gonadotrope cells of the anterior pituitary gland, may stimulate higher levels of gonadotropin production in lines of swine with increased ovulation rates. In the pig, the sequences for the genes encoding the subunits comprising the gonadotropins, FSH and LH (alpha-, FSHbeta- and LHbeta-sub-units), have recently been reported. In addition, the sequence for the porcine GnRH receptor gene was identified by researchers at the University of Guelph. Upon isolation of the gene, investigators at the USDA Meat Animal Research Center have determined that it is located in a similar region as a chromosomal marker for ovulation rate. Therefore, the GnRH receptor gene represents both a physiological and positional candidate for genes influencing ovulation rate in swine. Isolation of these sequences allows for quantification of GnRH receptor and gonadotropin subunit gene expression levels, so comparisons can be made between lines of pigs with ovulation rate differences.

To examine the role of these genes in determining ovulation rate between swine lines, we used swine lines with divergent ovulation rates. Females of the Chinese Meishan breed have a higher ovulation rate than occidental breeds, resulting in four to five more piglets per litter. Thus, Meishan pigs may harbor genetic differences with the potential to enhance reproductive performance of white crossbred pigs. Consistent with the Meishan model, researchers at UNL have developed a line of white crossbred pigs that were selected 11 generations for an index of ovulation rate and embryonic survival, followed by nine generations of selection for increased litter size. At generation 10, females from the UNL Index line ovulated 7.4 more eggs and at generation 19, produced 2.53 more live born piglets per litter than un-selected, control animals.

Materials and Methods: Animals and Treatments

All animal procedures conducted in this experimentation were approved by the UNL Institutional Animal Care and Use Committee. White crossbred Index and Control line gilts (Generation 23) were obtained from the University of Nebraska Swine Unit. Gilts of the Meishan breed were obtained from the United States Department of Agriculture, Roman L. Hruska U.S. Meat Animal Research Center in Clay Center, Neb. Gilts were housed in pens with a minimum of 8 square feet of floor space and received 4 pounds of feed per day with water available ad libitum. Estrous detection was initiated at 155 days of age for Index and Control gilts and at 95 days of age for Meishan gilts.

Upon completion of the third (Index and Control) or sixth (Meishan) estrus, 10 gilts from the Index and Control lines and 12 Meishan gilts were ovariectomized during the luteal phase (day 5 to 15 of the estrous cycle) to remove any confounding effects of steroid hormones (i.e., estrogen) on expression patterns of the genes of interest. During the third estrous cycle, Meishan gilts have similar ovulation rates to that of gilts from occidental breeds. Thus, Meishan females were ovariectomized during the sixth estrous cycle to assure that they would have increased ovulation rates than gilts from the Control line.

Eighteen days after ovariectomy, gilts from each line were randomly assigned to treatment groups and received an injection of either the specific GnRH antagonist, SB-75 (10 |ig biologically active compound per kg of body weight; UNL Protein Core Facility), or vehicle (0.9% saline) at 60, 36 and 12 hours prior to slaughter. The GnRH antagonist was used to block the effects of GnRH, which is dramatically increased in gilts following ovariectomy. Therefore, GnRH levels were expected to be elevated in ovariectomized gilts treated with vehicle and significantly reduced in ovariectomized gilts treated with the GnRH antagonist, SB-75.

Data Collection

Blood samples were collected prior to ovariectomy and the first treatment, as well as at slaughter. Following slaughter, the anterior pituitary gland was removed. Serum concentrations of FSH and LH were determined using a ra-dioimmunoassay validated in our laboratory. Levels of FSH and LH were determined using a known standard curve of the respective hormone, run simultaneously with the unknown samples. Total RNA, which was extracted from the anterior pituitary tissue, was converted to cDNA and used in quantitative real-time polymerase chain reaction assays to measure expression of the GnRH receptor, glycoprotein alpha-subunit, FSH-beta-subunit and LHbeta-subunit

Further Information

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Source: University of Nebraska, Lincoln - March 2006