Methyl donors dietary supplementation to gestating sows diet improves the growth rate of offspring and is associating with changes in expression and DNA methylation of insulin‐like growth factor‐1 gene

The study aimed to investigate the effects of maternal dietary methyl donors on the performance of sows and their offspring, and the associated hepatic insulin‐like growth factor‐1 (IGF‐1) expression of the offspring. A total of 24 multiparous sows were randomly fed the control (CON) or the CON diet supplemented with methyl donors (MD) at 3 g/kg betaine, 15 mg/kg folic acid, 400 mg/kg choline and 150 μg/kg VB₁₂, from mating until delivery. After farrowing, sows were fed a common lactation diet through a 28‐days lactation period and six litters per treatment were selected to be fed until at approximately 110 kg BW. Maternal MD supplementation resulted in greater birthweight (p < 0.05) and increased the piglet weights (p < 0.01) and litter weights (p < 0.05) at the age of day 28, compared with that in CON group. The offspring pigs in the MD group had greater ADG (p < 0.05) and tended to lower F:G ratio (p = 0.07) compared with that of CON group from day 28 to 180 of age. The offspring pigs from MD group had greater serum IGF‐1 concentrations and expressions of hepatic IGF‐1 gene and muscular IGF‐1 receptor (IGF‐1r) protein at birth (p < 0.05), and greater hepatic IGF‐1 protein (p = 0.03) and muscular IGF‐1r gene expressions (p < 0.05) at slaughter, than that from the CON group. Moreover, the methylation at the promoter of IGF‐1 gene in the liver of newborn piglets and finishing pigs was greater in the MD group than that of the CON group (p < 0.05). In conclusion, maternal MD supplementation throughout gestation could enhance the birthweight and postnatal growth rate of offspring, associated with an increased expression of the IGF‐1 gene and IGF‐1r, as well as the altered DNA methylation of IGF‐1 gene promotor.     

Proper reprogramming of imprinted and non‐imprinted genes in cloned cattle gametogenesis

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non‐imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non‐cloned bulls. We found no differences between cloned and non‐cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha‐satellite and Art2) in oocytes recovered from cloned and non‐cloned cows. Again, no significant differences were observed between clones and non‐clones. These results suggested that imprinted and non‐imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.     

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5‐methylcytosine‐5mC and 5‐hydroxymethylcytosine‐5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT‐♀ and SCNT‐♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT‐♀ embryos was greater than that of SCNT‐♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT‐♀ embryos, whereas 5hmC was majorly expressed in SCNT‐♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT‐♂ embryos were higher than those of SCNT‐♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight‐stage of the IVF, SCNT‐♀ and SCNT‐♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT‐♂ embryos at the eight‐cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two‐cell, eight‐cell and blastocysts of SCNT‐♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT‐♀ embryos than that of SCNT‐♂ embryos.     

Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.     

The rate and pattern of urea infusion into the rumen of wethers alters nitrogen balance and plasma ammonia

Changes in N balance, urinary excretion of purine derivative (PD), urea, creatinine and ammonia and plasma ammonia, glucose, urea, insulin and IGF‐1 were examined in four wethers (37 ± 2.6 kg BW). The animals were fitted with permanent ruminal catheters, fed lucerne hay (9.4 MJ/day; 23 g N/day; 7 g soluble N/day, 6 equal meals/day) and treated with contrasting rates of urea infusion into the rumen: first, a continuous infusion (CT), at 3.2 mg urea‐N/min for 10 days and then a discontinuous infusion (DT) at 156 mg urea‐N/min for 4 min; in 6 daily doses with the meals for 7 days. N balance was calculated from pooled samples of faeces and urine. Jugular blood samples were collected before and 1.5 h after the morning meal (M1) on days CT10, DT2, DT4 and DT6. N retention decreased during DT (p = 0.01) due to a significant increase of N excretion in urine (4 g/day; p = 0.009) and faeces (1 g/day; p = 0.02). Dry matter (p < 0.001) and N digestibility in vivo (p = 0.01) decreased significantly during DT. Urinary urea and PD excretion were not altered by treatment. Significant linear (p = 0.004) and quadratic (p = 0.001) effects were observed for plasma ammonia in M1 (from 170 CT10 to 235 μm DT2 and returned to 120 μm DT6). No changes were observed in plasma glucose, urea, insulin and IGF‐1. Results indicate that changes from CT to DT reduced N retention in sheep due to enhanced urinary N excretion, but it was not associated with changes in urinary urea or PD excretion; or plasma concentrations of insulin and IGF‐1. As the dry matter (DM) an N digestibility could account a 0.23 of the decrease in N retention; the largest fraction of the reduction in N retention remained unexplained by the results.     

Teat characteristics in relation to animal temperament during milking in buffaloes,and comparison of buffalo and cow teat morphology

This study aimed to examine the teat characteristics in relation to the animal temperament during milking in the Anatolian buffaloes using ultrasonographic, histomorphological and immunohistochemical methods. The teat canal length (TCL), teat wall thickness (TWT), teat cisternal diameter (TCD), teat diameter (TD), teat length (TL), and teat circumference (TC) values in docile (n = 5) and nervous (n = 7) buffaloes were measured at the 0th, 3rd and 6th minute of stimulation. In additional experiments, comparative histomorphology and immunohistochemical examinations of buffalo (n = 7) and cow teats (n = 8) were performed. It was determined that post-stimulation mean TCL values were significantly higher in nervous buffaloes than those of teats in docile buffaloes (p < .05). A significant positive correlation between TCD and TD, TL and TC in both docile and nervous buffaloes was noted (p < .05). Unlike nervous buffaloes where only 3/14 teat canals were open by 3rd minute of milking stimulation, almost all (9/10) teat canals were observed opened in docile buffaloes. There were fewer muscle but more collagen bundles in buffalo teats compared with cow teats. It seems that temperament of animal during milking effects the milking efficiency, and in nervous buffaloes, probably the stimulation alone may not be sufficient for opening of the teat canal and hence achieve complete milking.     

Elevated cyclin D1 expression is governed by plasma IGF‐1 through Ras/Raf/MEK/ERK pathway in rumen epithelium of goats supplying a high metabolizable energy diet

The objective of this study was to explore the underlying mechanism of insulin‐like growth factor 1 (IGF‐1)–caused cell proliferation of rumen epithelium in goats fed a high metabolizable energy (ME) diet. In this study, young goats were fed either a low ME [LL, n = 9, ME: 0.57 MJ/kg^0.75/day] or high ME [HL, n = 9, ME: 1.00 MJ/(kg^0.75/day)] diet for 42 day. The time duration of G₁‐phase was shortened as a result of enhanced expression of cyclin D1 mRNA in the HL group (p < 0.05). It was suggested that a high ME diet promoted cell transition from G₀/G₁ to S‐phase via cyclin D1. The level of phosphorylation of ERK was higher in HL than LL group (p < 0.05). In cell culture, the ERK was phosphorylated by IGF‐1 treatment. The proliferative effects of insulin‐like growth factor 1 (IGF‐1, 25 ng/ml) on [³H] thymidine (TdR) incorporation into DNA and on cyclin D1 protein expression of rumen epithelial cells were inhibited by PPP (the inhibitor of type 1 IGF receptor) (p < 0.05) and ERK inhibitor (p < 0.05) in vitro. Thus, IGF‐1 up‐regulated cyclin D1 expression and accelerated G₁‐phase progression in the cell cycle through Ras/Raf/MEK/ERK pathway in rumen epithelium of goats.     

Effects of Saccharomyces cerevisiae supplementation on growth performance,plasma metabolites and hormones,and rumen fermentation in Holstein calves during pre‐ and post‐weaning periods

This study aimed to evaluate the effects of supplementing Saccharomyces cerevisiae (SC) during the pre‐ and post‐weaning periods on growth, metabolic and hormonal responses, and rumen fermentation in calves. Three‐week‐old Holstein calves were assigned to either control (n = 12) or SC group (n = 12), the latter of which received 2 × 10⁹ cfu/day of SC. The experiment was conducted over a period of 7 weeks around weaning. Daily gain (DG) in the SC group was higher (p < .05) than that in the control group. In the SC group, plasma glucose, insulin, and growth hormone (GH) concentrations were higher (p < .05) and concentrations of glucagon and insulin‐like growth factor 1 (IGF‐1) tended to be higher (p < .1) than in the control group. Proportion of rumen propionate and concentration of rumen ammonia nitrogen at 10 weeks of age were greater (p < .05) in the SC group than that in the control group. Supplementation of SC around weaning may improve dietary nutrient and energy availability and increase plasma GH and IGF‐1 concentrations. These changes observed in SC‐supplemented calves could be closely related to the improvement of DG.     

Effects of Dietary Supplementation of β‐hydroxy‐β‐methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets

The effects of dietary β‐hydroxy‐β‐methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β‐hydroxy‐β‐methylbutyrate calcium (HMB‐Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor‐4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin‐like growth factor‐1 (IGF‐1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.     

DNA methylation patterns at the IGF2‐H19 locus in sperm of Swiss Landrace and Swiss Large White boars

DNA methylation patterns at the IGF2‐H19 locus were investigated in sperm DNA from Swiss Landrace (SL) and Swiss Large White (LW) boars. The putative IGF2 differentially methylated regions (DMR) 0, 1 and 2, a quantitative trait nucleotide (QTN) region in the intron 3 and a CpG island in the intron 4 of the IGF2 gene as well as three regions around porcine CTCF binding sites within the H19 differentially methylated domain (DMD) were selected for the DNA methylation analysis. In both breeds putative IGF2 DMR0, 1, 2 and H19 DMD were hypermethylated. Significant differences in DNA methylation content were found between the two breeds in the two DMD regions proximal to the H19 gene. The IGF2 QTN region and the CpG island in the IGF2 intron 4 were hypomethylated in sperm DNA of both breeds. The methylation analysis revealed significantly more methylated CpG sites in the intron 4 of sperm from the LW breed than in that from SL. No difference was found in global DNA methylation between the two breeds. These results indicate differences in DNA methylation patterns between breeds and it remains to be established whether variation in DNA methylation patterns impacts on phenotypic traits.     

Clinical efficacy of intrauterine cephapirin benzathine administration on clearance of uterine bacteria and subclinical endometritis in postpartum buffaloes

This study examined the effect of single IU administration of cephapirin on clinical recovery, clearance of uterine bacteria and reproductive performance of postpartum buffaloes with subclinical endometritis (SCE). Buffaloes (n = 86) at 35 days postpartum (DPP) with >10% polymorphonuclear (PMN) cells in endometrial cytosmears were designated as positive (SCEP, n = 29), and buffaloes with ≤10% PMN cell were designated as negative (SCEN, n = 57) for SCE. Out of 29 positive buffaloes, 15 were administered a single intrauterine dose of cephapirin benzathine on 40 DPP (SCEP‐CB), while the remaining 14 animals were kept as untreated control (SCEP‐C). All animals were observed regularly for oestrous signs and were again subjected to cytobrush sampling on the first postpartum (FPP) oestrus. Buffaloes positive for SCE at 35 DPP were later considered “recovered” if their PMN cells dropped to ≤5% on the FPP oestrus. Presence of Escherichia coli, Arcanobacterium pyogenes and Fusobacterium necrophorum in uterus was detected based upon PCR amplification of genes related to bacteria‐specific virulence factors. A total of 66.7% of SCEP‐CB group buffaloes recovered as compared to 28.6% in SCEP‐C (χ² = 4.21; p < 0.05). Rate of bacterial clearance did not differ between treated (38.5%) and untreated buffaloes (8.3%) (χ² = 1.67; p > 0.05). The median days to first service did not differ significantly (p > 0.05) among the three groups, whereas cephapirin administration reduced (p < 0.05) the days open by 14 days in SCEP‐CB compared to SCEP‐C buffaloes. SCEP‐CB buffaloes were as likely to conceive as SCEN, whereas SCEP‐C had 0.28 hazard ratio for pregnancy. In conclusion, a single treatment with cephapirin benzathine at 40 DPP improved the reproductive performance of buffaloes with subclinical endometritis.     

Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in cross‐bred buffaloes

This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross‐bred buffaloes. A total of 134 cross‐bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 10¹⁰ CFU/ml of the C501 (pVAX‐asd‐IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF_2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX‐asd‐IS) elicited significant immune responses, and anti‐inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti‐inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX‐asd‐IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross‐bred buffaloes.     

Associations of Leptin and Pituitary‐Specific Transcription Factor Genes’ Polymorphisms with Reproduction and Production Traits in Dairy Buffalo

This study aimed to detect the genetic variability in Leptin and Pit‐1 genes using polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing also to explore their possible associations with reproductive and productive traits of Egyptian buffaloes. Regarding Leptin gene, three genotypes (AA, AG and GG) were identified with frequency of 0.54, 0.40 and 0.06, respectively, and the genotypes were distributed according to the Hardy–Weinberg equilibrium. Allele A was comparatively higher than G with frequency of 0.74 and 0.26, respectively. For Pit‐1 gene, the association could not be performed due to the monomorphism (BB). The results showed that AA genotypes were found to be superior in most of production and reproduction traits. AA genotypes yielded more milk (2332.34 kg, p = 0.04) with higher fat% (6.10, p = 0.004) and fat yield (155.75 kg, p = 0.06), reach peak milk production at 42.19 days and required 2.19 services for conception in comparison with GG genotypes. Birthweight of animals with AA genotype was lesser than with GG genotype (39.35 and 43.67 kg, p = 0.02, respectively). The days open is numerically better in AA genotype animals (99.35 days), but the difference between the three genotypes was non‐significant. The distinct significant associations reported in this study suggested that Leptin is reputable candidate genetic marker, which might be used to enhance animals’ genetic potential for milk production in conjunction with reproduction.     

DNA methylation status in tissues of sheep clones

Genomic imprinting and DNA methylation play an important role in mammalian development. Many cloned animals showed heterogeneous DNA methylation profiles. However, there are fewer reports in cloned lambs because of a lack of genomic imprinting information. In this study, we investigated DNA methylation patterns in CpG islands and differentially methylated regions of putative imprinted gene Peg10 and imprinted genes Dlk1, Igf2R and H19 in cloned lambs. Five organs from two cloned lambs died shortly after birth and two normal controls were investigated. We observed normal DNA methylation profiles in cloned lambs. The imprinted genes Dlk1, Igf2R and H19 in livers, kidneys, hearts, muscles and lungs of the two cloned lambs exhibited relatively normal DNA methylation, except for Peg10 showing some differences between controls and cloned lambs. Our results indicate that somatic cell nuclear transfer-produced sheep exhibited relatively normal DNA methylation pattern and experienced normal DNA methylation reprogramming at imprinted loci.     

Effect of feeding rice whole crop silage on growth rate,levels of vitamin A, β‐carotene,vitamin E and IGF‐1 in plasma and skeletal muscle protein degradation in Japanese black calves

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, β‐carotene, vitamin E and IGF‐1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma β‐carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma β‐carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.     

Insulin‐Like Growth Factor‐1 Regulates the Expression of Luteinizing Hormone Receptor and Steroid Production in Bovine Granulosa Cells

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin‐like growth factor‐1 (IGF‐1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF‐1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF‐1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF‐1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non‐luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid‐sized follicles (6–8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF‐1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF‐1 (0.1–100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF‐1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF‐1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF‐1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.