Genome-Wide Association Studies for Methane Production in Dairy Cattle.

Genomic selection has been proposed for the mitigation of methane (CH4) emissions by cattle because there is considerable variability in CH4 emissions between individuals fed on the same diet. The genome-wide association study (GWAS) represents an important tool for the detection of candidate genes, haplotypes or single nucleotide polymorphisms (SNP) markers related to characteristics of economic interest. The present study included information for 280 cows in three dairy production systems in Mexico: 1) Dual Purpose (n = 100), 2) Specialized Tropical Dairy (n = 76), 3) Familiar Production System (n = 104). Concentrations of CH4 in a breath of individual cows at the time of milking (MEIm) were estimated through a system of infrared sensors. After quality control analyses, 21,958 SNPs were included. Associations of markers were made using a linear regression model, corrected with principal component analyses. In total, 46 SNPs were identified as significant for CH4 production. Several SNPs associated with CH4 production were found at regions previously described for quantitative trait loci of composition characteristics of meat, milk fatty acids and characteristics related to feed intake. It was concluded that the SNPs identified could be used in genomic selection programs in developing countries and combined with other datasets for global selection.

Genome-wide association study for milk somatic cell score in holstein cattle using copy number variation as markers.

Mastitis, the most common and expensive disease in dairy cows, implies significant losses in the dairy industry worldwide. Many efforts have been made to improve genetic mastitis resistance in dairy populations, but low heritability of this trait made this process not as effective as desired. The purpose of this study was to identify genomic regions explaining genetic variation of somatic cell count using copy number variations (CNVs) as markers in the Holstein population, genotyped with the Illumina BovineHD BeadChip. We found 24 and 47 copy number variation regions significantly associated with estimated breeding values for somatic cell score (SCS_EBVs) using SVS 8.3.1 and PennCNV-CNVRuler software, respectively. The association analysis performed with these two software allowed the identification of 18 candidate genes (TERT, NOTCH1, SLC6A3, CLPTM1L, PPARα, BCL-2, ABO, VAV2, CACNA1S, TRAF2, RELA, ELF3, DBH, CDK5, NF2, FASN, EWSR1 and MAP3K11) that result classified in the same functional cluster. These genes are also part of two gene networks, whose genes share the 'stress', 'cell death', 'inflammation' and 'immune response' GO terms. Combining CNV detection/association analysis based on two different algorithms helps towards a more complete identification of genes linked to phenotypic variation of the somatic cell count.

Increased body condition score through increased lean muscle, but not fat deposition, is associated with reduced reproductive response to oestrus induction in beef cows.

Energy reserve, estimated as body condition score (BCS), is the major determinant of the re-initiation of ovarian activity in postpartum cows. Leptin, IGF-I and insulin are positively related to BCS and are putative mediators between BCS and reproductive function. However, when BCS and body composition dissociates, concentrations of these metabolic hormones are altered. We hypothesized that increasing lean muscle tissue, but not fat tissue, would diminish the reproductive response to oestrus induction treatments. Thirty lactating beef cows with BCS of 3.10±1.21 and 75.94±12 days postpartum were divided in two groups. Control cows (n=15) were supplemented with 10.20 kg of concentrate daily for 60 days. Treated cows (n=15) were supplemented equally, and received a ß-adrenergic receptor agonist (ß-AA; 0.15 mg/kg BW) to achieve accretion of lean tissue mass and not fat tissue mass. Twelve days after ending concentrate supplementation/ß-AA treatment, cows received a progestin implant to induce oestrus. Cows displaying oestrus were inseminated during the following 60 days, and maintained with a fertile bull for a further 21 days. Cows in both groups gained weight during the supplementation period (Daily weight gain: Control=0.75 kg v. ß-AA=0.89 kg). Cows treated with ß-AA had a larger increase in BCS (i.e. change in BCS: control=1 point (score 4.13) v. ß-AA=2 points (score 5.06; P0.05) did not differ between groups. However, the number of cows displaying oestrus (control 13/15 v. ß-AA 8/15; P<0.05) and the percentage cycling (control 6/8 v. ß-AA 3/10; P=0.07) after progestin treatment and the pregnancy percentage at the end of the breeding period (control 13/15 v. ß-AA 8/15; P<0.05) were lower in ß-AA than control cows. In summary, the increase BCS through muscle tissue accretion, but not through fat tissue accretion, resulted in a lower response to oestrus induction, lower percentage of cycling animals and lower pregnancy percentage after progestin treatment; which was associated with a decrease in serum concentrations of leptin and IGF-I.

Reduced response to an estrous induction program in postpartum beef cows treated with zilpaterol and gaining body weight.

In beef cows, reduced energy intake delays first ovulation postpartum and is associated with lesser insulin, IGF-I and leptin concentrations. However, the close relationship among these hormones mask their individual roles in the reinitiation of ovarian activity. A ß-adrenergic receptor agonist (ßAR) was used to increase body condition score (BCS) and yet reduce body fat and leptin serum concentration to determine the specific role of leptin in the postpartum ovarian activity. Beef cows (n=77) with BCS 3.1 ± 1.4 received 2 kg/day of feed containing 0 or 0.15 mg/kg of zilpaterol (a synthethic ßAR), for 33 days. Estrus was induced with a progestin implant applied for 9 d and cows in estrus were bred by artificial insemination (AI). Zilpaterol administration increased (P<0.05) daily weight gain, muscle depth and BCS, with no changes in back fat depth, reducing fat to muscle ratio (P<0.05). At the time of AI, insulin (38%) and IGF-I (26%) concentrations were less in zilpaterol-treated cows (P<0.05), but leptin concentration was unaffected. Ovulation rate and animal with luteal activity after estrus induction were also reduced by 35% (P=0.05) and 56.5% (P=0.007), respectively, in zilpaterol-treated cows. Logistic regression estimates for BCS (P=0.016) and IGF-I concentration (P=0.03) were positively related with the occurrence of luteal activity. In addition, whilst back fat (P=0.009) had a positive effect on luteal activity, leptin concentration did not show a significant relationship. In conclusion, despite an increase in body weight and a positive change in BCS, the reduction in insulin and IGF-I concentrations, associated with ßAR treatment, reduced the response to induction of estrus. However only IGF-I, but not leptin or insulin, significantly influenced the odds for the occurrence of luteal activity after estrous induction in cattle with poor BCS.

Progesterone and the distribution of pituitary gonadotropin isoforms in cattle.

The objective of the present study was to determine the relative proportion of gonadotropin isoforms in bovine pituitary glands affected by progesterone. Twelve postpubertal heifers (Swiss-Zebu) were assigned to three groups (n=4): intact animals in the luteal phase of the estrous cycle (diestrus group); ovariectomized heifers with (OVXP) or without progesterone treatment (OVX). Prior to pituitary gland collection, a blood sample was taken from each animal to determine the circulating progesterone concentration. Pituitary protein extractions processed by chromatofocusing were eluted with a pH gradient ranging from 10.5 to 3.5. The LH and FSH eluent was grouped on the basis of the following three criteria: (1) as either a basic (pH>or=7.5), neutral (pH 7.4-6.5) and acid (pHor=10.5-3.5); (3) on the basis of distinct isoforms 12 peaks of which (A-L) were identified for LH and 11 (I-XI) for FSH. The analysis by range of pH and by pH of elution in the OVX and OVXP groups showed no difference in the LH and FSH isoform ratio, but diestrus cattle differs having a greater ratio (p<0.05) of basic LH isoforms (87.5+/-0.4%) and lesser ratio (p<0.05) of acid isoforms (5.4+/-0.7%). In the diestrus group, the ratio of acid FSH isoform increased (62.1+/-1.7%), while neutral isoforms decreased (5.7+/-0.4%, P<0.05). The analysis by isoform type of LH revealed a greater proportion of isoforms C (pH 9.4) and E (pH 9.0) in the groups with circulating progesterone when compared to the OVX group. The heterogeneity of FSH was quantitatively similar in most isoforms in the three groups, with the exception of the predominant isoform (VIII, pH 4.9) that was more abundant in the diestrus group (p<0.05). These results indicate that progesterone with other gonad factors influence the pituitary glicosylation altering the relative proportions of gonadotropin isoforms.

Stereological study of postnatal testicular development in Blackbelly sheep.

The objective was to characterize testicular development in Blackbelly sheep, focusing primarily on Sertoli cell number. Lambs (n=43) were allotted into eight groups, and surgically castrated at 0, 3, 6, 9, 12, 15,18 or 21 weeks of age (n=4-6 lambs per group). Testes were fixed and paraffin-embedded, cross-sections (5 microm) were stained and evaluated with quantitative morphometry techniques. Testis weight increased at a greater rate between 9 and 15 weeks of age, which was associated with remarkable changes in testicular histology, including increases in tubular tissue volume, and tubule diameter and length. Spermatogenesis started in a period between 9 and 12 weeks, lumen and elongated spermatids were observed for the first time at 12 weeks (78% of the tubules) and 15 weeks (37% of the tubules), respectively. Total number of Sertoli cells (mean+/-S.E.M.) increased steadily from birth (531+/-76 x 10(6)) to 15 weeks (12,008+/-1722 x 10(6)), with no changes afterwards. Sertoli cell number per gram of testicular tissue decreased as lambs were older, with the most remarkable change occurring between Weeks 9 and 12. An early increase in serum LH was observed at 6 weeks of age, with testosterone (T) increasing at Weeks 12 and 21. In conclusion, Sertoli cells maintained the capacity of proliferating from birth to 15 weeks of age in Blackbelly sheep; furthermore, the period of accelerated testis growth was associated with increased serum T concentration and with important changes in testicular morphology, as a consequence of the beginning and establishment of spermatogenesis and Sertoli cell maturation.

Luteinizing hormone (LH) isoforms in ruminants: characterization and physiological relevance.

The luteinizing hormone (LH) is secreted in multiple molecular forms into the blood stream; however, few studies have characterized the pattern of the circulating LH isoforms in domestic animals during different physiological stages. Most of the publications are related to the pattern of isoforms present in the anterior pituitary gland. This review includes recent evidence concerning the distribution of LH isoforms in the pituitary gland and serum in ruminants. The structural heterogeneity of this hormone is emphasized, including the glycosylation biosynthetic pathway, as well as the different proportions of oligosaccharides that confer particular functional characteristics to the heterodimer. Evidence for a regulating role of GnRH, estradiol and progesterone is discussed.

Caprine luteinizing hormone isoforms during the follicular phase and anestrus.

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 microg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH> or =7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH< or =6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p<0.05) in A (12.0+/-0.8%) as compared with F (5+/-2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH> or =10), which amounted to a percentage of 6.0+/-0.4% of the total observed during A, and 3+/-1% during F (p<0.05). Predominant isoforms in A eluted in the pH range 9.99-9.0 (42+/-3%) as compared to 7+/-3% (p<0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99-8.0, representing 55+/-8%, while in A the proportion was 11+/-2% (p<0.01). Isoforms eluted at the pH range 7.9-7 represented a significantly greater proportion during A (5.0+/-0.6%) as compared with F (3+/-1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

Ovine serum and pituitary isoforms of luteinising hormone during the luteal phase.

The relative abundance of the different isoforms of pituitary and circulating luteinising hormone (LH) in ewes, at different times after the administration of gonadotrophin-releasing hormone (GnRH), during the luteal phase of the oestrous cycle was investigated. Sixteen ewes on Day 9 of their cycle were divided into four groups (n = 4). The control group (T0) received saline solution; the remaining animals received 100 microg GnRH (i.m.) 30, 90 or 180 min (T30, T90 and T180, respectively) before serum and pituitary gland collection. Luteinising hormone polymorphism was analysed by chromatofocusing (pH 10.5-3.5). The LH eluted from each chromatofocusing was grouped on the basis of the following three criteria: (1) according to the pH of elution (pH > or = 10-3.5); (2) as either a basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic (pH < or = 6.4) elution of LH of serum and hypophyseal origin; and (3) on the basis of distinct isoforms, of which 10 (A-J) were identifiable in hypophyseal extracts and four (A-D) were found in the serum. In general, the most abundant forms of LH in both the pituitary and serum, at all times, were basic. However, that proportion was greater in hypophyseal extracts (84 +/- 3%, 81 +/- 4%, 82 +/- 3% and 83 +/- 2% at T0, T30, T90 and T180, respectively) than in serum (51 +/- 5%, 48 +/- 10% and 54 +/- 6% at T30, T90 and T180, respectively). Neutral and acidic LH made up a larger proportion of the total LH in sera (neutral: 17 +/- 4%, 20 +/- 6% and 23 +/- 3% at T30, T90 and T180, respectively; acidic: 32 +/- 8%, 32 +/- 11% and 23 +/- 6% at T30, T90 and T180, respectively) than in the pituitary extracts (neutral: 4.0 +/- 0.7%, 10 +/- 4%, 7 +/- 2% and 5.0 +/- 0.5% at T0, T30, T90 and T180, respectively; acidic: 12 +/- 3%, 11 +/- 2%, 12 +/- 2% and 12 +/- 2% at T0, T30, T90 and T180, respectively) at all times. These data reveal that the relative composition of the LH present in the pituitary gland and the LH secreted into the circulation is different, with more neutral and acidic isoforms being secreted. The pattern of circulating LH isoforms changes between 30 and 180 min after GnRH peak induction, with a greater proportion of isoform C (eluting between pH 7.0 and 6.5) at T180 compared with T30 and T90.

Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers.

The pattern of distribution of circulating luteinizing hormone (LH) isoforms in cattle during estrus and the luteal phase was investigated. In each stage, the stage of the estrous cycle was synchronized in seven Holstein heifers with a prostaglandin analogue. After estrus was detected, blood samples were taken at 2-h intervals for 24h. In the luteal phase, animals received 250 microg i.v. of GnRH and blood samples were collected every 15 min for 5h. LH concentration in the samples was determined. Samples with the greatest LH concentration in estrus (pre-ovulatory peak) and those collected 60 min after GnRH administration (luteal phase) were analyzed by chromatofocusing, eluted with a pH gradient from 10.5 to 3.5. Eluted LH was grouped into basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH < or = 6.4) as well as by pH unit. In both phases, basic forms were the most abundant, and these were greater (P < 0.05) during the luteal phase (78.4 +/- 4.2%) as compared with during estrus (57.1 +/- 6.2%); the proportion of neutral and acidic isoforms in estrus (13.7 +/- 2.6%; 28.5 +/- 2.8%) was greater (P < 0.05) as compared with the luteal phase (3.0 +/- 0.7; 18.7 +/- 3.4). These results indicate that the relative proportion of LH isoforms secreted by the adenohypophysis differ by stage of estrous cycle. The addition of excess of NaCl to the column modifies the antigen-antibody binding in the RIA, and the proteins eluted are erroneously quantified as LH; this is an artifact of the technique.