In a previous study, we showed that access to willow fodder decreased somatic cell counts (SCC) in the milk of local Mamber goats grazing in brushland at the end of lactation. To test whether the consumption of willow affects the cells of the immune system, Alpine crossbred dairy goats grazing in the same environment were either offered free access to freshly cut willow fodder (W, n = 24) or not (C, n = 24) for 2 weeks. The willow fodder contained 7.5 g/kg DM of salicin. The other major secondary compounds were catechin, myricitrin, hyperin and chlorogenic acid (2.2, 2.6, 1.0 and 0.75 g/kg DM, respectively). Udder health status was determined before the experiment, and each of the two groups included five (W) or six (C) goats defined as infected, as established by microbial cfu in milk, and 19 (W) or 18 (C) non-infected goats. Goats ingested, on average, 600 g of DM from willow (25% of food intake), resulting in minor changes in dietary quality compared to the controls, as established by faecal near-IR spectrometry. Throughout the 2 weeks of experiment, differences between groups in dietary CP contents were minor and affected neither by infection nor by access to willow; the dietary percentage of neutral detergent fibre (NDF) decreased in C and increased in W; dietary acid detergent fibre (ADF) increased; and the dietary tannin contents decreased for both treatments. However, milking performance and milk quality attributes in both W and C goats were similar. Initial SCC and milk neutrophil (cluster of differentiation (CD)18+ and porcine granulocyte (PG)68) cell counts were higher in infected than in non-infected goats; counts decreased significantly in W but not in C uninfected goats. The percentage of CD8+ T-cells increased in all C goats, while in the W group, a significant increase was found only for infected goats. The consumption of willow mitigated an increase in CD8+ in blood and triggered an increase in CD8+ in milk, suggesting an immune-regulatory effect independent of udder status. To our knowledge, this is the first report of a direct nutraceutical effect of fodder ingestion on the immune status of goats.

In semiarid conditions, feed is often scarce and variable with underfeeding being common; these factors can potentially induce fertility reductions in both sexes. Sexually active bucks are able to very efficiently fertilize out-of-season goats, but we do not know whether underfeeding would reduce the ability of bucks to fertilize goats during these periods. Two experiments were conducted to determine (i) testicular size and change of odor intensity of undernourished bucks exposed to long days and (ii) the ability of these bucks to stimulate reproductive activity in seasonally anestrous goats. In experiment 1, bucks (n = 7) were fed 1.5 times the normal maintenance requirements from September to May and formed the well-fed group. Another group of bucks (n = 7) were fed 0.5 times the maintenance requirements and formed the undernourished group. All bucks were subjected to artificially long days from 1 November to 15 January; this period was followed by a natural photoperiod until 30 May. Body weight, scrotal circumference and male odor intensity changes were determined every 2 weeks. In experiment 2, two groups of female goats (n = 26 each) were exposed to well-fed (n = 2) or undernourished bucks (n = 2) on 31 March. Ovulations and pregnancy rates were determined by transrectal ultrasonography. In experiment 1, a treatment by time interaction was detected for BW, scrotal circumference and odor intensity changes (P < 0.001). The BWs of well-fed bucks were greater than those of the undernourished bucks from October to May (P < 0.01), as were the scrotal circumferences from December to March (P < 0.05) and odor intensities from February to May (P < 0.05). In experiment 2, the proportions of females that ovulated at least once (100% v. 96%) or those that were diagnosed as pregnant (85% v. 77%; P > 0.05) did not differ significantly between the goats exposed to well-fed or undernourished bucks. The interval between the introduction of bucks and the onset of estrous behavior was shorter in goats exposed to well-fed bucks compared to the interval for those goats exposed to undernourished bucks (2.5 ± 0.2 v. 9.5 ± 0.6 days; P < 0.05). We conclude that undernourishment reduces the testicular size and odor intensity responses in bucks exposed to long days, but that undernourished bucks are still able to stimulate reproductive activity in seasonally anestrous goats, as is also the case for well-fed bucks.

The objectives of the research described here were to describe the persistence of intramammary infections (IMI) caused by coagulase negative staphylococci (CNS) in goats using strain-typing, and to evaluate the relationship between species-specific CNS IMI and somatic cell score (SCS) at the udder-half level. Udder-half milk samples were collected from all 909 lactating goats (1817 halves; 1 blind half) in a single herd. Milk samples were cultured on Columbia blood agar, and 220 goats with at least one half yielding a single colony type CNS were enrolled for two additional half-level samplings at approximately 1-month intervals. Isolates were identified to the species level by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry or PCR amplification and partial sequencing of tuf or rpoB. An IMI was defined as persistent when ≥1 follow-up sample yielded the same species and strain as on Day 0 based on pulsed-field gel electrophoresis. A generalised mixed linear model was used to evaluate the odds of persistence as a function of CNS species. A mixed linear model was used to evaluate the relationship between IMI status on a given day and SCS. Among 192 IMI, 69.8% were persistent based on species and strain-type. Staphylococcus simulans IMI had higher odds of persistence than Staphylococcus arlettae IMI. In primiparous goats, Staphylococcus epidermidis IMI was associated with higher SCS than S. arlettae, Staphylococcus xylosus and ‘other CNS’ IMI. The differences detected in the present study between CNS species, with regard to persistence of IMI and association with SCS, highlight the need to study CNS at the species and strain level to understand the pathogenicity and epidemiology of CNS in goats.

Nutrition is a major factor that regulates ruminant milk components, particularly its fatty acid (FA) composition, which is an important determinant of milk nutritional quality. In the mammary gland, milk component biosynthesis involves a large number of genes under nutritional regulation that are not well understood. Thus, the objective of the present study was to evaluate the effects of extruded linseeds (EL) alone or in combination with fish oil (ELFO) on goat mammary gene expression. In total, 14 goats were fed one of the following three diets: a natural grassland hay basal diet (CTRL) alone, CTRL supplemented with 530 g/day of EL, or 340 g/day of EL plus 39 g/day of fish oil (ELFO). Mammary secretory tissues were collected after slaughter on day 28, to determine the expression of 14 lipogenic genes and five lipogenic enzyme activities and transcriptomic profiles. The mRNA abundance decreased for SCD1 (P<0.1) with ELFO v. CTRL, and for AZGP1 (P<0.1) and ACSBG1 (P<0.05) decreased with EL v. ELFO and the CTRL diets (only for ACSBG1), respectively. Transcriptomic analyses performed using a bovine microarray revealed 344 and 314 differentially expressed genes (DEG) in the EL and ELFO diets, respectively, compared with the CTRL diet, with 76 common DEGs. In total, 21 and 27 DEGs were involved in lipid metabolism and transport class in the EL and ELFO v. the CTRL diets, respectively, with eight common genes (ALDH3B1, ALDH18A1, DGKD1, ENPP1, IL7, NSMAF, PI4KA and SERINC5) down-regulated by these two treatments. In EL v. CTRL diets, a gene network related to lipid metabolism and transport was detected. Although this network was not detected in the ELFO v. CTRL analysis, five genes known to be involved in lipid metabolism and transport were up-regulated (SREBF1, PPARG and GPX4) or down-regulated (FABP1 and ENPP6) by ELFO. The protein metabolism and transport biological processes were largely altered by both EL and ELFO v. CTRL diets without changes in major milk protein secretion. Amino acid metabolism was highlighted as an enriched network by Ingenuity Pathway Analysis and was similar to cellular growth and proliferation function. Two regulation networks centered on the estrogen receptor (ESR1) and a transcriptional factor (SP1) were identified in EL and ELFO v. CTRL diets. In conclusion, these results show that these two supplemented diets, which largely changed milk FA composition, had more effects on mRNA linked to protein metabolism and transport pathways than to lipid metabolism, and could affect mammary remodeling.

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus–oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants.

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

Goat breeds from subtropical latitudes show different annual reproductive cycles. Some of them display large seasonal variations in their annual breeding season, while others display a moderate seasonality or sexual activity all year round. This reproductive seasonality causes seasonality of milk, cheese and meat productions and, as a consequence, induces wide variation in producer incomes. To solve this problem and provide methods allowing producers to breed animals during the anestrous period and stabilize production all year round, it is necessary to have a deep knowledge of their annual sexual activity and to identify the environmental factors controlling the timing of the annual reproductive cycle. Then, it is possible to build on these knowledge sustainable breeding techniques adapted to the environmental, economic and social characteristics of the local breeding system. In this review, I will illustrate this strategy through the example of our experiments in subtropical goats. First, we determined the characteristics of the annual breeding season in both male and female goats. Second, we identified the photoperiod as the major environmental factor controlling the timing of this annual breeding season. Third, we used the photoperiod to stimulate indirectly the sexual behavior of does. Indeed, we used photoperiodic treatments to stimulate the sexual activity of bucks during the non-breeding season. These sexually active male goats were then used to induce and synchronize the estrous behavior and ovulatory activity of anestrous females in confined or grazing conditions by using the ‘male effect’. Under subtropical conditions, these results constitute an original manner to control the reproductive activity of local goats using the photoperiod combined with the ‘male effect.’

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2–6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM 199 supplemented with 10 μg each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5°C) for 4.5 h during transportation. Then, oocytes were transferred into 75 μl of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5°C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37°C water bath for 15s. Motile fractions were selected by swim-up, then incubated for 90 mm in TALP with 10 μg heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 μl) were added to 10 μl medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM 199 + 20% FBS at 37°C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 μl of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Bovine and caprine milks have a similar overall gross composition, but vary considerably in the ratios of their casein components. These differences cause significant changes in the ability of caseins to bind and stabilize calcium (Ca). It might be expected that these in vitro variations, which are thought to be due to differences in casein phosphopeptides (CPP) content, could lead to in vivo differences in the digestion and absorption of Ca. To test this hypothesis three milks with different casein ratios [bovine (B), caprine high in αs1-casein (CH) and caprine low in αs1-casein (CL)] were compared with regard to Ca absorption and deposition in growing male rats. For comparison, each milk was Ca-fortified (BCa-milk, CHCa-milk, and CLCa-milk) and CPP, prepared by enzymatic hydrolysis from the respective caseins (extrinsic CPP), were added to both native and Ca-milks. The effects of added CPP (extrinsic) could then be compared with intrinsic CPP released from the gastrointestinal digestion of caseins. Total gastric Ca was sampled at 15, 30 and 60 min after ingestion. No differences were found among the native milks with or without CPP, but the Ca from all Ca-milks (regardless of casein type) appeared to clear the stomach more rapidly and this was enhanced by the extrinsic CPP. The total intestinal Ca was not different among the native milks±CPP, however, it rose more rapidly with Ca fortification, and was higher at 30 min for all CPP-Ca-milks. At 60 min the total intestinal Ca level fell for the CPP-Ca-milks while all others continued to rise. These observations suggest that the CPP in Ca-milks enhance gastric clearance and uptake from the intestine. Ca availability from BCa-milk, CHCa-milk, and CLCa-milk with and without CPP was estimated by both plasma and femur uptake of 45Ca. Ca availability was enhanced at 5 h in the plasma in each case by added CPP. In all cases CPP stimulated Ca availability in the femur, but the CL-CPP was higher (P<0·05) than that of either CH-CPP or B-CPP (extrinsic CPP). Based on the results of this study we can conclude that the addition of CPP will have beneficial effect on the absorption of Ca in growing rats from CaCO3 added to bovine and caprine milks.

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM – control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

β-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the β-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

Xanthine oxidoreductase (XOR) was purified from goats' milk. The u.v.–visible absorption spectrum was essentially identical to those of the corresponding bovine and human milk enzymes and showed an A280/A450 ratio of 5·20±0·12, indicating a high degree of purity. Like bovine and human milk XORs, enzyme purified from goats' milk showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150000. On Western blotting, mouse monoclonal anti-human XOR antibody cross-reacted with purified caprine and bovine XORs. The specific xanthine oxidase activity of goats' milk XOR, however, was very much lower than that of bovine XOR, although NADH oxidase activities of XOR from the two sources were similar. In these respects, the caprine milk XOR mirrors the human milk enzyme, in which case the kinetic effects have previously been attributed to relatively low molybdenum content. The molybdenum content of goats' milk XOR also was shown to be relatively low, with 0·09 atoms Mo per subunit, compared with 0·55 atoms Mo per subunit for the bovine enzyme. A parallel purification of human milk XOR showed 0·03 atoms Mo per subunit. The possible physiological significance of the low molybdenum content of the caprine milk enzyme and of its correspondingly low enzymic activity is discussed.