Production of a gonadotropin-releasing hormone 2 receptor knockdown (GNRHR2 KD) swine line.

Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we reported that GNRH2 and GNRHR2 mediate LH-independent testosterone secretion from porcine testes. To further explore this ligand-receptor complex, a pig model with reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentiviral particles were generated and microinjected into the perivitelline space of zygotes, and embryos were transferred into a recipient. One GNRHR2 knockdown (KD) female was born that subsequently produced 80 piglets from 6 litters with 46 hemizygous progeny (57% transgenic). Hemizygous GNRHR2 KD (n = 10) and littermate control (n = 7) males were monitored at 40, 100, 150, 190, 225 and 300 days of age; body weight and testis size were measured and serum was isolated and assayed for testosterone and luteinizing hormone (LH) concentrations. Body weight of GNRHR2 KD boars was not different from littermate controls (P = 0.14), but testes were smaller (P < 0.05; 331.8 vs. 374.8 cm3, respectively). Testosterone concentrations tended (P = 0.06) to be reduced in GNRHR2 KD (1.6 ng/ml) compared to littermate control (4.2 ng/ml) males, but LH levels were similar (P = 0.47). The abundance of GNRHR2 mRNA was reduced (P < 0.001) by 69% in testicular tissue from mature GNRHR2 KD (n = 5) versus littermate control (n = 4) animals. These swine represent the first genetically-engineered model to elucidate the function of GNRH2 and its receptor in mammals.

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element.

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.

Production of bovine alpha-lactalbumin in the milk of transgenic pigs.

High production of milk and its components are necessary to allow maximal growth of developing pigs. In this study, transgenic pigs were produced containing the alpha-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein alpha-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine alpha-lactalbumin gene. The gene construct contained 2.0 kb of 5' flanking region, the 2.0 kb coding region, and 329 bp of 3' flanking region. Sows hemizygous for the transgene produced as much as .9 g of bovine alpha-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total alpha-lactalbumin concentration of pig milk throughout a lactation. The concentration of bovine alpha-lactalbumin was highest on d 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine alpha-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on d 0 of lactation, but by d 20 of lactation the ratio was .43 to 1. This suggested that the bovine transgene and the endogenous porcine gene are under slightly different control mechanisms. The higher level of total alpha-lactalbumin present on d 0 of lactation was correlated with higher lactose percentage on d 0 in transgenic sows (3.8%), compared with controls (2.6%) (P < .01). Although there was also a trend for higher lactose percentage in transgenic sows on d 5 and 10 of lactation, no significant differences were observed. These data suggest that alpha-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of alpha-lactalbumin early in lactation may boost milk output.

Growth and body composition of Meishan and Yorkshire barrows and gilts.

Five barrows and five gilts of each of two breeds (Meishan [Ms] and Yorkshire [Y]) were slaughtered at birth and at 41, 71, 123, and 171 d of age and five gilts of each breed were slaughtered at 260 d of age. Major organ and visceral weights were obtained immediately postmortem (PM), whereas carcass characteristics, carcass composition, femur measurements, and two individual muscle weights were obtained 24 h PM. Linear and quadratic regression coefficients on age differed between the two breeds, in favor of Y barrows and gilts, for live weight, carcass weight, longissimus muscle area (LMA), liver, heart, spleen, and kidney weights, and femur cross-sectional, medullary, and cortical areas (P < .001), leaf fat weight, and percentage of body fat and protein. In contrast to somatic tissue, Ms gilts had uteri and ovaries that grew faster than those of Y gilts from birth to 260 d of age (P < .05), although the uterus and ovary weights were similar for both breeds by 260 d of age (P < .05). Regression coefficients differed (P < .05) between the two sexes for live, carcass, liver, lung and trachea, stomach and esophagus and leaf fat weights, dressing percentage, percentage of body protein, 10th rib backfat (TRBF) thickness (P < .001), and small intestine, kidney (P < .01), heart, and spleen weights (P < .05). Breed differences in regression coefficients differed between the two sexes for percentage of body protein and leaf fat weights (P < .05). Yorkshire pigs were larger, later-maturing pigs that grew faster from birth to 171 d of age. Yorkshire pigs slaughtered at 171 d of age had heavier total wholesale cut (WC), trimmed cut (TC), and boneless cut (BC) weights than did Ms pigs of the same age (P < .001).

Comparison of the semen characteristics of Fengjing, Meishan and Yorkshire boars.

Semen characteristics of Chinese Fengjing, CHinese Meishan and American Yorkshire boars were examined. Samples were collected from 24 boars: 6 Fengjing, 12 Meishan and 6 Yorkshire. Three semen characteristics and 6 biochemical evaluations of semen or seminal plasma were analyzed. The whole semen parameters measured were gelatinous (gel) and gel-free volume and progressive motility. Fengjing boars were higher in gel volume than Meishan (P < 0.05) but not Yorkshire boars (P > 0.25), while Yorkshire boars were higher than Meishan boars in gel volume (P < 0.10). The gel-free volume was higher in Yorkshire and Fengjing boars than Meishan boars (P < 0.10), but Fengjing gel-free volumes did not differ from Yorkshire gel-free volumes (P > 0.80). However, the only difference detected for progressive motility was between Fengjing and Meishan boars (78.5 vs 74.5%; P < 0.10). Sperm concentration was higher in Meishan than Yorkshire boars (P < 0.01) although these breeds did not differ from Fengjing boars (P > 0.18). The biochemical messurements made were fructose, galactose, inositol, total carbohydrate and total protein in seminal plasma and pH in gel-free semen. Fengjing boars had higher concentrations of galactose in seminal plasma than Meishan or Yorkshire boars (P < 0.05), while Meishan seminal plasma had higher galactose concentrations than Yorkshire seminal plasma (P < 0.10). Fructose, inositol and total carbohydrate concentrations were all higher in Fengjing and Meishan seminal plasma than Yorkshire seminal plasma (P < 0.05). Fengjing seminal plasma had higher levels of inositol than Meishan seminal plasma (769 vs 566 mg/100 ml; P < 0.10). Furthermore, Fengjing and Meishan semen had similar fructose and total carbohydrate concentrations (P > 0.80). The pH level and total protein concentration did not differ among the 3 breeds (P > 0.30). This study indicated that breed differences were detected for several seminal parameters, although no single breed had consistantly higher values for all the parameters measured.

Effects of porcine somatotropin on growth and carcass composition of Meishan and Yorkshire barrows.

Effects of porcine somatotropin (pST) on growth and carcass characteristics of Meishan (Ms) and Yorkshire (Y) barrows given 3 mg of pST or saline daily were determined for two end points. First, 26 Ms and 26 Y barrows were treated from 119 d of age until the Y barrows reached 108 kg. Second, another 18 Ms barrows were treated to 108 kg BW. Age- and weight-matched treatments were analyzed. Results for both groups indicated pST effects (P < .05) for feed conversion (+) and intake (-), dressing percentage (-), percentage of carcass fat (-) and protein and water (+), leaf fat (-), backfat (BF) thickness (-), longissimus muscle area (LMA; +), skin thickness (+), muscle firmness and marbling scores (-), organ weights (+), belly (-), clear plate (-), ham (+), and total boneless cuts (BC; +). Somatotropin effects were also present for loin (-) and boneless Boston butt (BBB; +) in the age-matched group and for ADG (+), carcass weight (-), loin (-), jowl (-), and tenderloin (+) in the weight-matched group. Breed effects (P < .05), in favor of Y barrows, in both treatments existed for ADG (+) and feed intake (+), carcass weight (+), dressing percentage (+), LMA (+), skin thickness (-), muscle color and firmness scores (-), muscling score (+), all wholesale cuts (WC; +) except clear plate (age-matched), all trimmed cuts (TC; +) except picnic shoulder (weight-matched), and all BC (+). Breed effects, in favor of Y barrows, were also determined for carcass length (+), percentage of carcass ash (-), leaf fat (+), average BF thickness (+), and heart (-) and liver (-) weights in age-matched animals and percentage of carcass fat (-), protein (+), water (+), leaf fat (-), 10th rib, average, and P2 BF thicknesses (-), marbling score (-), femur length (-), and liver weights (+) in weight-matched animals. A higher response to pST (P < .05) was determined in Ms barrows than in Y barrows for percentage of carcass protein (+), liver (+), and heart (+) in the age-matched treatment and 10th rib BF thickness (-) and heart weight (+) in the weight-matched treatment. Yorkshire barrows treated with pST had more improved values for color score (+; age-matched) and BBB (+; weight-matched).

Age at puberty, ovulation rate, uterine length, prenatal survival and litter size in Chinese Meishan and Yorkshire females.

Studies on the ovulation rate, prenatal survival and litter size of Chinese Meishan pigs have given widely divergent results depending on the extent of inbreeding of the animals, their original genetic diversity, the age and parity, and the conditions of management. To obtain meaningful results, it is necessary to characterize the population under study. The following report characterizes populations of Meishan and Yorkshire of a widely diverse background. First farrowing data were collected on 21 Meishan and 20 Yorkshire gilts. Meishan gilts had 12.4 fully formed piglets and Yorkshire gilts had 7.4 fully formed piglets (P < 0.01). Meishan gilts averaged 1.86 mummified fetuses per litter vs 0.05 per Yorkshire litter (P < 0.01). Yorkshire piglets averaged 1.3 kg body weight at birth vs 0.9 kg for Meishan piglets (P < 0.01). At 47 days of second gestation, 19 Meishan and 12 Yorkshire sows averaged 22.7 and 16.3 corpora lutea (CL), respectively (P < 0.01). Uterine length and number of fetuses were not different (P > 0.40) in the two breeds. Daily estrous detection of 50 Meishan and 34 Yorkshire gilts began at 60 and 120 days of age, respectively. Meishan gilts reached sexual maturity at 95 days of age, which was 105 days earlier than Yorkshire gilts (P < 0.01). Meishan gilts were in estrus nearly 1 day longer than Yorkshire gilts at first, second and third estrus (P < 0.05). No differences in cycle length between breeds were detected for the first or second estrous cycle (P > 0.60). Nineteen Meishan gilts were slaughtered at 51 days of gestation and their reproductive tracts were recovered. The mean number of dissected CL (17.0), number of fetuses (13.1), total uterine length (396 cm), spacing per fetus (29.9 cm), allantoic (124.9 ml) and amniotic (32.2 ml) volumes, crown-rump length (82.8 mm), weight (35.4 g), sex, and direction of each fetus were determined. Chinese Meishan gilts reached puberty much earlier and were in estrus longer than Yorkshire gilts and Meishan sows had more CL than Yorkshire sows.