The importance of small ruminants to the dairy industry has increased in recent years, especially in developing countries, where it has a high economic and social impact. Interestingly and despite the fact that the mammary gland is the specialised milk production organ, very few authors studied the modifications occurring in the mammary gland through the lactation period in production animals, particularly in the small ruminants, sheep (Ovis aries) and goat (Capra hircus). Nevertheless, understanding the different mammary gland patterns throughout lactation is essential to improve dairy production. In addition, associating these patterns with different milking frequencies, lactation number or different diets is also of high importance, directly affecting the dairy industry. The mammary gland is commonly composed of parenchyma and stroma, which includes the ductal system, with individual proportions of each changing during the different periods and yields in a lactation cycle. Indeed, during late gestation, as well as during early to mid-lactation, mammary gland expansion occurs, with an increase in the number of epithelial cells and lumen area, which leads to increment of the parenchyma tissue, as well as a reduction of stroma, corresponding macroscopically to the increase in mammary gland volume. Throughout late lactation, the mammary gland volume decreases owing to the regression of the secretory structure. In general, common mammary gland patterns have been shown for both goats and sheep throughout the several lactation stages, although the number of studies is limited. The main objective of this manuscript is to review the colostrogenesis and lactogenesis processes as well as to highlight the mammary gland morphological patterns underlying milk production during the lactation cycle for small ruminants, and to describe potential differences between goats and sheep, hence contributing to a better description of mammary gland development during lactation for these two poorly studied species.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Maintaining metabolic balance is a key factor in the health of dairy cattle during the transition from pregnancy to lactation. Little is known regarding the role of the circadian timing system in the regulation of physiological changes during the transition period. We hypothesized that disruption of the cow's circadian timing system by exposure to chronic light-dark phase shifts during the prepartum period would negatively affect the regulation of homeostasis and cause metabolic disturbances, leading to reduced milk production in the subsequent lactation. The objective was to determine the effect of exposure to chronic light-dark phase shift during the last 5 wk prepartum of the nonlactating dry period on core body temperature, melatonin, blood glucose, β-hydroxybutyric acid (BHB) and nonesterified fatty acid (NEFA) concentrations, and milk production. Multiparous cows were moved to tiestalls at 5 wk before expected calving and assigned to control (CTR; n = 16) or phase-shifted (PS; n = 16) treatments. Control cows were exposed to 16 h of light and 8 h of dark. Phase-shifted cows were exposed to the same photoperiod; however, the light-dark cycle was shifted 6 h every 3 d until parturition. Resting behavior and feed intake were recorded daily. Core body temperature was recorded vaginally for 48 h at 23 and 9 d before expected calving using calibrated data loggers. Blood concentrations of melatonin, glucose, BHB, and NEFA were measured during the pre-and postpartum periods. Milk yield and composition were measured through 60 DIM. Treatment did not affect feed intake or body condition. Cosine fit analysis of 24-h core body temperature and circulating melatonin indicated attenuation of circadian rhythms in the PS treatment compared with the CTR treatment. Phase-shifted cows had lower rest consolidation, as indicated by more total resting time, but shorter resting period durations. Phase-shifted cows had lower blood glucose concentration compared with CTR cows (4 mg/mL decrease), but BHB and NEFA concentrations were similar between PS and CTR cows. Milk yield and milk fat yield were greater in PS compared with CTR cows (2.8 kg/d increase). Thus, exposure to chronic light-dark phase shifts during the prepartum period attenuated circadian rhythms of core body temperature, melatonin, and rest-activity behavior and was associated with increased milk fat and milk yield in the postpartum period despite decreased blood glucose pre-and postpartum. Therefore, less variation in central circadian rhythms may create a more constant milieu that supports the onset of lactogenesis.

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Since lactation is one of the most metabolically demanding states in adult female mammals, beautifully complex regulatory mechanisms are in place to time lactation to begin after birth and cease when the neonate is weaned. Lactation is regulated by numerous different homeorhetic factors, all of them tightly coordinated with the demands of milk production. Emerging evidence support that among these factors are the serotonergic and circadian clock systems. Here we review the serotoninergic and circadian clock systems and their roles in the regulation of mammary gland development and lactation physiology. We conclude by presenting our hypothesis that these two systems interact to accommodate the metabolic demands of lactation and thus adaptive changes in these systems occur to maintain mammary and systemic homeostasis through the reproductive cycles of female mammals.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

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The aim of this study was to investigate the BW and immune status of lambs reared under natural conditions or under artificial conditions fed two different colostrum amounts. In this study, 60 lambs were randomly divided into groups according to treatment. Twenty lambs remained with their dams (natural rearing (NR) group). Forty lambs were removed from their dams at birth. Lambs were bottle-fed with a pool of sheep colostrum, receiving either 4 g of IgG/kg of BW at birth (C4 group) or 8 g of IgG/kg of BW at birth (C8 group). The total colostrum amount was equally divided into three meals at 2, 14 and 24 h after birth. After this period, lambs were bottle-fed a commercial milk replacer. Blood plasma sample analysis and BW recordings were carried out before feeding at birth and then at 1, 2, 3, 4, 5 and 20 days after birth. Another blood sample analysis and BW recording was carried out when animals reached 10 kg of BW. During weaning (30 days), sampling was carried out every 5 days. Blood plasma was used to determine the concentrations of IgG and IgM and the complement system activity -total and alternative pathways. The NR group showed greater BW than the C4 and C8 groups during milk feeding period, whereas the C4 and C8 groups had greater BW than the NR group at the end of weaning period. The C8 and NR groups had greater plasma IgG and IgM concentrations than the C4 group during milk feeding period. In addition, C4 and C8 groups showed similar IgG concentrations and greater IgM concentrations than the NR group at the end of the weaning period. Complement system activity was greater in the NR group than in the C4 and C8 groups during the first 3 days after birth. In conclusion, lambs fed amounts of colostrum equivalent to 8 g of IgG/kg of BW showed similar immune variables compared to lambs reared under natural conditions, obtaining a greater BW at the end of the weaning period. Nevertheless, this study shows that not only the colostrum amount but also the management during the milk feeding and weaning period, such as stress produced by dam separation, milk quality and suckling frequency, can affect the final immune status of lambs.Keywords: immune, lamb, colostrum, weaning, artificial rearing ImplicationsColostrum plays a fundamental role in the immunization of newborn ruminants. Artificial rearing is a common method used in dairy farms, increasing the amount of milk for processing. The aim of this study was to feed newborn lambs colostrum with two different IgG concentrations and to compare these animals to natural dam fed lambs. This study showed that administration of 8 g of immunoglobulin g/kg BW at birth, equally divided into three colostrum meals (2, 14 and 26 h), achieved higher circulating immunoglobulin G concentration when compared to the natural rearing treatment, although both artificial rearing groups decreased BW after birth. IntroductionThe relationship between colostrum and the survival of newborn ruminants has been long characterized (Argüello et al., 2004;Castro et al., 2011;Abdou et al., 2014)...