Further testing of Melatonin Receptor 1a for out-of-season reproduction in the Cornell flock and allelic frequencies compared with Romney sheep.

Sheep are seasonally polyestrous breeders, meaning they breed when day length shortens in the autumn. Ewes respond to changing day length through chemical pathways involving melatonin receptors. Some breeds, such as Dorset, are known to be less seasonal with many ewes able to breed and lamb year-round. The Melatonin Receptor 1a () gene was identified as a candidate gene controlling out-of-season lambing. The first studies in the Cornell STAR accelerated lambing flock found that a allele was associated with a shorter time to first lambing and a shorter period between lambings. The favorable allele was denoted the allele and the unfavorable allele, the allele. This study evaluated additional data for the effect of the polymorphism on sheep reproduction. Genotypic frequencies among the 320 sheep in this study differed between Romney and breeds selected for accelerated lambing ( < 0.02), but they did not deviate from Hardy-Weinberg equilibrium. Using 228 ewes from the Cornell University flock, we found no association ( > 0.05) between the allele and success for out-of-season lambing and no significant differences were observed in several production measures, such as number of lambs delivered per yr or number of lambs weaned ( > 0.05). With few ewes in the flock, these results may be due to the high level of selection for accelerated lambing. This study shows that the allele may not be a beneficial marker for use within flocks seeking to improve production and the ability to lamb out-of-season. These findings warrant additional research on the genetics of aseasonality in sheep.

Effects of service sire on prenatal mortality and prolificacy in ewes.

Ability to select service sires that minimize partial or complete losses of pregnancy could have major economic impacts in sheep production systems. This study tested the null hypothesis that survival of potential progeny did not vary with breed type of service sire or among individual rams. Data included 980 ewes on 10 farms; each ewe was pregnant to 1 of 67 rams of 12 breeds. Number of conceptuses was estimated once during pregnancy by ultrasonography, either transrectal (embryos) or transabdominal (fetuses), and was compared with number of lambs born to estimate losses. Data were examined first for number of lambs born and second for documented losses. Individual service sires affected number born (P < 0.001), which varied from 0.70 to 2.45 lambs per pregnant ewe. The main effects of breed type on lambs born were not significant, but breed types of both service sires (P < 0.0002) and ewes (P < 0.001) interacted with diagnosed number of conceptuses. Lambs born varied with ewe age (P < 0.0001) and among farms (P < 0.0001), and statistically, farms interacted with number of diagnosed conceptuses (P < 0.0001); season had no effect. In documented losses, there were both main effects of individual service sire and a service sire × number of diagnosed embryos interaction (P < 0.005). Thus, ewes bred to some rams were more apt to lose single pregnancies, whereas ewes bred to other rams were more apt to lose 1 or more embryos or fetuses from multiple pregnancies. Breed type of service sire affected (P < 0.05) prenatal death. Complete losses of single conceptuses tended to be greater in ewes bred to black-faced or hair-type rams (service sire breed type × number of diagnosed conceptuses; P < 0.09). Breed type of ewes also varied in incidence of complete losses (P < 0.05); hair-type ewes (46%) lost more (P < 0.02) documented conceptuses from examination to birth than black-faced (27%), white-faced (20%), or dairy-type (25%) ewes. Greater losses of singles than of multiples occurred in black-faced (37% vs. 18%) and hair-type (64% vs. 27%) ewes than in other breeds (ewe breed type × number of conceptuses; P < 0.03) per ewe. Surprisingly, purebred conceptuses were lost less often (24%) than crossbreds (36.4%; P < 0.002). Selection of rams based on records of prenatal losses in ewes they serviced may be a method to decrease embryonic and fetal wastage. However, further study to determine repeatability of differences among service sires from year to year will be required.

Genetic mapping of quantitative trait loci for milk production in sheep.

A backcross pedigree using dairy East Friesian rams and non-dairy Dorset ewes was established specifically to map quantitative trait loci (QTL) affecting milk production in sheep. Ninety nine microsatellite markers of an initial set of 120 were successfully genotyped and informative on 188 animals of this backcross pedigree. Test-day milk records on individual ewes were used to estimate several milk yield related traits, including peak milk yield and cumulative milk yield to 50 (MY50), 100 (MY100) and 250 days (MY250). These traits, as well as estimated breeding value of backcross ewes extracted from the genetic evaluation file of the entire flock, were used in interval mapping. Ovine chromosomes 2, 12, 18, 20 and 24 were identified to harbour putative QTL for different measures of milk production. The QTL on Ovis aries chromosomes (OAR) 2 and 20 mapped to locations where similar trait QTL have already been mapped in other studies, whereas QTL on OAR 12, 18 and 24 were unique to our backcross pedigree and have not been reported previously. In addition, all identified QTL regions were syntenic with bovine chromosomal segments revealed to harbour QTL affecting milk production traits, providing supporting evidence for the QTL identified here.

Genetic mapping of quantitative trait loci for aseasonal reproduction in sheep.

The productivity and economic prosperity of sheep farming could benefit greatly from more effective methods of selection for year-round lambing. Identification of QTL for aseasonal reproduction in sheep could lead to more accurate selection and faster genetic improvement. One hundred and twenty microsatellite markers were genotyped on 159 backcross ewes from a Dorset × East Friesian crossbred pedigree. Interval mapping was undertaken to map the QTL underlying several traits describing aseasonal reproduction including the number of oestrous cycles, maximum level of progesterone prior to breeding, pregnancy status determined by progesterone level, pregnancy status determined by ultrasound, lambing status and number of lambs born. Seven chromosomes (1, 3, 12, 17, 19, 20 and 24) were identified to harbour putative QTL for one or more component traits used to describe aseasonal reproduction. Ovine chromosomes 12, 17, 19 and 24 harbour QTL significant at the 5% chromosome-wide level, chromosomes 3 and 20 harbour QTL that exceeded the threshold at the 1% chromosome-wide level, while the QTL identified on chromosome 1 exceeded the 1% experiment-wide significance level. These results are a first step towards understanding the genetic mechanism of this complex trait and show that variation in aseasonal reproduction is associated with multiple chromosomal regions.

Effect of prolactin, beta-lactoglobulin, and kappa-casein genotype on milk yield in East Friesian sheep.

The effect of prolactin (PRL), beta-lactoglobulin (beta-LG), and kappa-casein (CSN3) on milk yield was estimated in an East Friesian dairy sheep population from Old Chatham Sheepherding Company, New York. Genotypes were determined by PCR amplification followed by digestion with HaeIII and RsaI for PRL and beta-LG, respectively, and by PCR amplification for CSN3. Monthly milking records and pedigree information were used to evaluate the effect of each polymorphism on milk yield. Results indicated that PRL genotype had a significant effect on milk yield. Ewes carrying one A allele produced 110.6g more milk per day than ewes with no A alleles. There was no statistical difference between ewes with only one A allele and ewes with 2 A alleles. No association among polymorphisms at the beta-LG and CSN3 loci and milk yield was found. The results presented in this study indicate that the PRL gene is a potential marker that could be used in selection programs for improving milk yield in dairy sheep.

Association between melatonin receptor 1A gene polymorphism and reproductive performance in Dorset ewes.

The response to melatonin expression is one way that circadian rhythms of many biological processes are regulated. To evaluate the relationship between the melatonin receptor 1A (MTNR1A) gene and reproductive performance, records were compared in Dorset and 3/4-Dorset x 1/4-East Friesian ewes expressing different genotypes at the MTNR1A gene in the Cornell University sheep flock. There were 116 ewes with first lambing records, consisting of 91 Dorset and 25 crossbred ewes. Of these, 104 ewes had second lambing records. Genotypes were determined by PCR amplification of a fragment of the ovine MTNR1A gene followed by digestion with MnlI and RsaI restriction enzymes. The effects of breed, year of birth, season of birth or season of first conception, and each polymorphism on days to first lambing and days between first and second lambings were evaluated. Our results show that ewes with at least 1 M allele are able to conceive at younger ages, better able to breed and conceive out-of-season, and have shorter intervals between first and second lambings than ewes expressing only the m allele. The results presented in this study show, for the first time, an association of the MTNR1A gene and lambing frequency and confirm the importance of the MTNR1A gene as a potential DNA marker for out-of-season breeding.

Antibody and skin-test responses of sheep vaccinated against Johne's Disease.

Current vaccines against Mycobacterium avium subsp. paratuberculosis (MAP, Johne's Disease) may cause animals to react positively when tested for Mycobacterium bovis (Bovis). Therefore, the effects of vaccination on MAP serum Ab and skin-test responses to MAP and Bovis PPD were compared in 25 ewes vaccinated against MAP with 24 control ewes in an infected flock 3 years post-vaccination. MAP-specific Ab levels were higher (P<0.001) in vaccinated ewes than in control ewes. All increases in skinfold-thickness from 0 to 48h were greater (P<0.0001) than zero while increases in skinfold-thickness from 48 to 72h were greater (P<0.05) than zero for Johnin but not for Bovis PPD. The Vaccine x PPD x Time interaction for skinfold-thickness was significant (P<0.001) with greater increases to Johnin than to Bovis, but with much greater increases in vaccinated ewes. These data suggest that administration of vaccines against MAP developed from whole organisms increase the likelihood that animals will be classified as "responders" to a Bovis screening test and negative by the follow-up comparative cervical tuberculin test, but they also show that vaccination initiates both humoral and cell-mediated MAP-specific responses.

Effect of testosterone on insulin-like growth factor-I, androgen receptor, and myostatin gene expression in splenius and semitendinosus muscles in sheep.

Testosterone is known to act differentially on skeletal muscle from different regions. Two genes likely to mediate the testosterone effect are IGF-I, an important growth regulator acting in an autocrine and paracrine way, and androgen receptor (AR), as receptor density could account for differential muscle growth. Another muscle-specific gene that may play a role in differential muscle growth is myostatin, a member of the transforming growth factor-beta superfamily, shown to be a negative regulator of skeletal muscle mass. The objective of this study was to quantify and compare the expression of these three genes in two different skeletal muscles in sheep. East Friesian x Dorset-sired ram lambs from Dorset ewes were used in a 2 x 4 factorial experiment. Eighteen sets of twins were assigned to four age groups corresponding to 77, 105, 133, and 161 d of age, and one individual from each set was castrated at birth. Total RNA was extracted from samples of splenius (SP) and semitendinosus muscles collected at the time of slaughter. Insulin-like growth factor-I mRNA was measured using competitive reverse-transcription PCR. Androgen receptor and myostatin mRNA were measured by ribonuclease protection assay with standard curves. Weight of SP was greater than semitendinosus in rams compared with wethers at 105, 133, and 161 d (P = 0.05, P = 0.04, and P = 0.02, respectively). The difference in IGF-I mRNA levels between the two muscles was greater in rams than in wethers at 133 (P = 0.001) and 161 d (P = 0.014), and the difference in AR mRNA levels was greater in rams than in wethers at 105, 133, and 161 d (P = 0.002, P < 0.001, and P < 0.001, respectively), with greater abundance in the SP. No difference was found in myostatin mRNA level between the two muscles in rams and wethers at any age. These results suggest that locally produced IGF-I and the regulation of AR expression are important for sexually dimorphic muscle growth patterns.

The effect of milk intake on forage intake and growth of nursing calves.

Thirty-nine Holstein steer calves were assigned to one of five treatments at birth and individually fed for 200 d with milk replacer reconstituted to equal the fat and protein concentration of beef cow milk. Treatment levels were the quantities of reconstituted milk fed per day based on lactation curves, which were based on peak milk levels (PML) of 2.72, 5.44, 8.16, 10.88, and 13.6 kg/d, respectively. In addition to reconstituted milk, chopped alfalfa hay was offered ad libitum to allow for maximal voluntary forage consumption. All calves were fed a high-energy diet postweaning until they reached a similar degree of fatness in the 12th rib (4 to 5% chemical fat) as determined by ultrasound. There were differences (P < 0.05) among groups in weaning weight, preweaning ADG, age, and weight at slaughter. During the preweaning phase, there was a linear relationship (P < 0.01) for daily milk and forage DE intake; however, DE intake per unit of BW did not differ across treatments (P = 0.06). Increasing PML resulted in a linear (P < 0.01) decrease in alfalfa hay intake in the preweaning phase, and G:F increased quadratically (P < 0.01). During the postweaning phase, preweaning milk intake had no meaningful effect on postweaning ADG, but overall ADG had a linear relationship (P < 0.01) with preweaning milk level. There was no effect of PML on the 12th-rib lipid percent, marbling score, or quality grade, but protein and fat concentration in the carcass and empty BW increased linearly (P < 0.01) with PML. The group fed at 2.72 kg/d PML was 58 kg lighter (P = 0.03) and required 34 d more (P < 0.01) to reach the predetermined degree of fatness at slaughter than the group fed at 13.6 kg/d PML, suggesting that increased milk production by the dam can decrease the number of days to the slaughter weight at which a similar rib lipid concentration is reached.

Source and level of supplemental protein for growing lambs.

Two 3 x 2 factorial growth trials and a companion metabolism trial with 13, 15, or 17% dietary CP (DM basis), with or without 3% of the DM replaced with slowly degraded menhaden fish meal, were conducted to determine if level of dietary protein influences whether slowly degraded protein improves lamb growth and protein use. The growth trials included 32 and 34 pens of two weanling lambs initially weighing 23 to 26 kg and fed for 42 d. The metabolism trial included 12 additional lambs fed in metabolism cages with a 2-wk adjustment period, a 1-wk preliminary period, and a 7-d collection period. Plasma urea N (PUN) was measured in all lambs at the conclusion of the second growth trial and at the end of the metabolism trial. There was a protein level x protein source interaction (P = 0.05) for PUN of the 12 lambs in the metabolism trial but not for the 68 lambs in the second growth trial. Replacement of part of the soybean meal protein with protein from fish meal did not affect ADG or G:F at any protein level, but it lowered (P = 0.08) PUN in the second growth trial. Plasma urea N values were higher (P = 0.002) in lambs fed diets with 15 or 17% CP; however, ADG (P = 0.037 in Exp. 1 and P = 0.055 in Exp. 2), and G:F (P = 0.094 in Exp. 1 and P = 0.003 in Exp. 2) were lower for lambs fed the diets with 13% CP. There was little difference in ADG or G:F between lambs fed the diets with 15 or 17% CP, suggesting that a CP level of 15% with supplemental protein from soybean meal would be optimal for 25- to 40-kg growing Finnsheep x Dorset lambs.

Gene expression in sexually dimorphic muscles in sheep.

Testosterone is known to act differentially on skeletal muscle from different regions of the body. Two genes likely to mediate the testosterone effect are insulin-like growth factor I (IGF-I), an important growth regulator acting in an autocrine and paracrine way, and androgen receptor (AR), because receptor density could account for differential muscle growth. Another muscle-specific gene that may play a role in differential muscle growth is myostatin, a member of the transforming growth factor-beta superfamily, shown to be a negative regulator of skeletal muscle mass. The objective of this study was to quantify and compare the steady state expression of these three genes in two different skeletal muscles in sheep. Eleven Dorset rams were slaughtered after reaching puberty and total RNA was extracted from samples of semitendinosus and splenius muscles. Insulin-like growth factor I mRNA was measured using a competitive reverse-transcription-polymerase chain reaction. Androgen receptor and myostatin mRNA were measured by a ribonuclease protection assay (RPA) with standard curves. The means (attomoles/microg RNA) for splenius and semitendinosus muscles were 1.39 and 1.02 (SE = 0.14), 4.05 and 2.96 (SE = 0.24), and 4.30 and 3.85 (SE = 0.37) for IGF-I, AR, and myostatin, respectively. The difference between the two muscles was significant for IGF-I and AR mRNA levels with higher levels in the splenius but not significant for myostatin. Our results show that locally produced IGF-I and the regulation of AR expression may be important for sexually dimorphic muscle growth patterns.

Phenotypic and phylogenetic characterization of ruminal tannin-tolerant bacteria.

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.

Vaccination schedules to raise antibody concentrations against epsilon-toxin of Clostridium perfringens in ewes and their triplet lambs.

The objective of this experiment was to compare vaccination schedules for ewes and their lambs to raise antibody concentrations to epsilon-toxin of Clostridium perfringens, the causative agent of enterotoxemia. Half of 200 Finnsheep x Dorset ewes were vaccinated with C. perfringens type D toxoid vaccine 3 wk before lambing. Serum samples were obtained from 20 ewes that were to be vaccinated and 20 ewes that would remain unvaccinated before treatment and at wk 2, 1, and 0 before the start of lambing. Antibody concentrations in sera of unvaccinated ewes remained at 2 IU/mL, but they peaked in vaccinated ewes at 15 IU/mL by wk 1 before lambing. Lambs from each of the first 13 and the first 14 sets of triplets from vaccinated and unvaccinated ewes, respectively, received one of three vaccination treatments: no vaccine (control), vaccination on d 1 and 21 of age, or vaccination on d 21 and 42 of age. Antibody concentrations declined in sera of vaccinated ewes from 8.5 IU/mL immediately after lambing to 3 IU/mL 12 wk later. Vaccination of lambs did not increase sera antibody concentration. However, prepartum vaccination of ewes significantly increased lamb antibody concentrations (19 IU/mL) compared with lambs reared by unvaccinated ewes (2 IU/mL). Vaccination of ewes resulted in lambs with higher antibody concentrations until wk 10 postpartum. Concentrations declined to .6 IU/mL in all lambs at 12 wk. Because concentrations of .2 IU/mL may be protective, these results indicate that vaccination of ewes before lambing imparts passive protection in lambs to 12 wk of age, whereas vaccination of young lambs provides no added protection.

Effect of testosterone on differential muscle growth and on protein and nucleic acid concentrations in muscles of growing lambs.

Growth, nucleic acid, and protein concentrations were measured in three muscles of 20 rams, 20 wethers, and 20 wethers implanted with testosterone. Two lambs from each group were slaughtered at 14-d intervals from 49 to 133 d, and then at 28-d intervals until 217 d, for a total of 10 slaughter ages. Immediately after slaughter, the semitendinosus, splenius, and triceps brachii muscles were removed, trimmed of adhering fat, and weighed. The DNA, RNA, and protein concentrations of these muscles were determined. Testosterone increased combined weight of the three muscles. The splenius muscles of rams and wethers implanted with testosterone were heavier and had a biphasic growth pattern as the combined muscle weight increased, whereas the splenius muscle of wethers had a single growth phase. Rams and implanted wethers had greater splenius muscle DNA and RNA concentrations than wethers as muscle weight increased. This model could be used to study the gene regulation of testosterone-induced muscle growth with the possibility of invoking similar effects in more economically important muscles.

Ontogeny of growth hormone, insulin-like growth factor-I, estradiol and cortisol in the growing lamb: effect of testosterone.

Exogenous sex steroids have altered growth hormone secretion in some domestic species. This study examined whether different physiological concentrations of testosterone alter muscle growth in sheep through modification of the somatotropic axis. The effects of testosterone on growth hormone (GH), insulin-like growth factor-I (IGF-I), estradiol (E2) and cortisol concentrations in growing lambs were evaluated in 20 rams, 20 wethers and 20 wethers receiving subcutaneous testosterone replacement therapy. Two animals from each of the three testosterone status groups were slaughtered at 14-day intervals from 49 to 133 days of age, and then at 28-day intervals until 217 days of age for a total of 10 slaughter ages. Animals were sampled every 10 min for an 8-h period 1 day prior to slaughter to characterize the episodic patterns of GH and testosterone. Immediately after slaughter, the semitendinosus, splenius and triceps brachii muscles were removed, trimmed of adhering fat and connective tissue, and weighed. Testosterone increased the combined muscle weight. GH concentrations decreased during the course of the experiment. However, there was no effect of testosterone on GH mean, baseline, amplitude or GH pulse frequency measured by PULSAR. IGF-I concentrations increased in response to testosterone treatment. Testosterone had no effect on cortisol levels while E2 levels were increased after 133 days. Increased muscle growth due to testosterone appeared to be caused either by a direct effect or by increased levels of IGF-I independent of circulating GH concentrations.

Effects of testosterone on skeletal growth in lambs as assessed by labeling index of chondrocytes in the metacarpal bone growth plate.

The effects of testosterone on the epiphyseal growth plate of metacarpal bones of growing sheep were evaluated in 20 rams, 20 wethers, and 20 wethers receiving subcutaneous testosterone replacement therapy. Two animals from each testosterone treatment group were slaughtered at 14-d intervals from 49 to 133 d, and then at 28-d intervals until 217 d, for a total of 10 slaughter ages. Immediately after slaughter, the cannon bones were dissected of extraneous tissue, weighed, and their lengths measured. Growth plates from the metacarpal bones were isolated and explants were cultured for 24 h in medium containing [3H]thymidine. After autoradiography, labeling index was calculated as the ratio of labeled to total nuclei in the resting and proliferative zones of the growth plate. Testosterone increased (P < .03) weight and length of the metacarpal bone. Increased bone length due to testosterone was associated, in part, with a higher (P < .05) labeling index in chondrocytes of the proliferative zone of the growth plate. Labeling indices in the resting zone chondrocytes of rams were higher near the time of puberty. Accelerated growth followed by cessation of growth occurs concurrently with puberty in males of several species and is accompanied by an increase in the blood concentration of testosterone. Testosterone may mediate this accelerated growth by first increasing bone growth and then depleting the source of stem cells in the cartilage growth plate, the site where growth in length of long bones occurs.

Technical note: chemical composition of Angus and Holstein carcasses predicted from rib section composition.

Seventy-four small-framed Angus and sixty-nine Holsteins were used to evaluate the classic Hankins and Howe equations for predicting percentages of water, protein, and lipid of steer carcasses varying in weight and breed type. Animals were fed either corn grain or corn silage diets, housed inside or outside, and slaughtered at one of five predetermined weights within breed. Soft tissues from a carcass side and the from the 9-10-11th rib section were separated from bone, and the percentages of water, protein, and lipid were measured. The Hankins and Howe equations predicted .81 +/- .181 percentage units more (P < .001) water and .99 +/- .213 percentage units less (P < .001) lipid than was measured in the carcasses. The residual difference for protein depended on breed and diet (P < .028) and decreased .22 +/- .078 percentage units for each percentage unit increase in predicted protein. During development of equations from our data, no statistical difference was found between Angus and Holstein in the relationship of carcass water to rib water. The relationship of carcass protein to rib protein had a lower (P < .1) intercept with a steeper (P < .055) slope for Angus than for Holstein steers, whereas for lipid there was no difference between the slopes, but Holsteins had one percentage unit more (P < .038) carcass lipid than Angus at the same percentage rib lipid. These results confirm that composition of the 9-10-11th rib section can be used accurately to predict carcass composition with minor adjustments for breed type.