J. Anim Sci. 2008. 86:10-14. doi:10.2527/jas.2007-0311
© 2008 American Society of Animal Science
Effects of short day photoperiod on prolactin signaling in dry cows: A common mechanism among tissues and environments?1,2
G. E. Dahl3
Department of Animal Sciences, Institute of Food and Agricultural Sciences, University of Florida, Gainesville 32611
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Abstract
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Photoperiodic manipulation has dramatic physiological and production effects in dairy cows. During lactation, exposure to long day photoperiod (LDPP) increases milk yield and circulating IGF-I and prolactin (PRL) concentrations. Conversely, cows housed under a short day photoperiod (SDPP) during the dry period produce more milk in the subsequent lactation than cows exposed to LDPP or natural photoperiod. Exposure to SDPP depresses PRL secretion but increases PRL receptor mRNA levels in mammary, immune, and hepatic tissues. In dry cows under SDPP, PRL signaling is a potential mechanism to drive more extensive mammary cell differentiation and growth relative to LDPP. In mammary biopsies taken during the dry period and into lactation, the amount of IGF-II mRNA was greater in SDPP vs. LDPP cows during the dry period, whereas IGFBP-5 mRNA increased in both groups during lactation even though photoperiodic treatments ended at parturition and all cows were on an ambient lighting schedule when lactating. Levels of IGF-I mRNA did not differ over time or between treatments; however, during the dry period, lower IGFBP-5 and increased IGF-II expression in SDPP cows may enhance mammary cell growth and survival. Key among the potential modulators of PRL signaling is the suppressors of cytokine signaling (SOCS) family. Mammary transcription of mRNA for SOCS proteins was low during the dry period but increased in lactation. During the dry period, SOCS mRNA level in the mammary gland of cows on SDPP was reduced compared with cows on LDPP, which may enhance PRL-induced proliferation and subsequent milk production. However, improved mammary capacity and immune function alone are likely insufficient to support increased milk yield. Using improved milk yield as a functional indicator of greater animal well-being during the transition, it is clear that some metabolic accommodation is necessary for expression of that capacity. Emerging evidence supports a link between PRL signaling and hepatic lipid metabolism, with decreases in PRL being beneficial to lipid metabolism. Extending that concept to broad environmental responses, it can be speculated that altered PRL signaling impairs lipid metabolism, mammary growth, and immune function under conditions of stress (e.g., heat stress) also. Thus, shifts in gene expression related to PRL signaling may provide an environmentally mediated mechanism to alter production and health in cows as they transition into lactation.
Key Words: photoperiod • prolactin • suppressors of cytokine signaling • environment
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INTRODUCTION
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Twenty-five percent of the variation in milk yield is attributable to genetics, whereas the remaining 75% is due to environmental factors (Bourdon, 1997
). Given the progress that was made through intensive genetic selection targeting only 25% of the total variation, the potential to dramatically increase milk yield and production efficiency through understanding and manipulating environmental factors is readily apparent. It is clear that photoperiod, the duration of light exposure animals receive on a daily basis, affects performance in cattle. This paper will briefly review the endocrine and production responses to photoperiod and then focus on tissue and cellular aspects of mammary development during the dry period of cows in response to different lighting schemes. Those data, with an emphasis on a prolactin (PRL)-mediated sequence of events, led to the development of a hypothesis that circulating PRL acts as a general environmental signal that alters events at multiple tissues in support of lactation. If correct, that hypothesis would have an effect on recommendations for management of light exposure and temperature of dry cows to optimize production and health.
It is important to begin with a brief review of terminology and physiology associated with photoperiodic responses. Photoperiod is defined as the duration of light exposure within a 24-h day. A long day or long day photoperiod (LDPP) consists of a 16- to 18-h period of light exposure. In contrast, a short day or short day photoperiod (SDPP) is characterized by 8 h of light followed by 16 h of darkness. Light and dark elicit contrasting effects on the secretion of melatonin from the pineal gland, such that circulating melatonin is virtually absent during light exposure but rapidly increases upon exposure to darkness (Dahl et al., 2000). It is the melatonin pattern, specifically the relative durations of elevated concentrations (i.e., presence or absence), that drives differential secretion of other hormones to influence circadian and seasonal processes. Of particular interest to this discussion is the effect of photoperiod on circulating concentrations of IGF-I and PRL, because both hormones influence function of the mammary gland as well as other tissues.
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PHOTOPERIOD EFFECTS ON LACTATION
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Although the most common adaptive response of animals to photoperiod relates to seasonal reproductive activity (Tucker and Ringer, 1982), that effect has been selected against in dairy cows. However, the final phase of reproduction, being lactation, remains responsive to photoperiodic variation. For example, LDPP increases milk yield in lactating cows, whereas exposure of dry cows to SDPP is associated with improved yield in the subsequent lactation (Dahl and Petitclerc, 2003). The milk yield response to LDPP during lactation averages around 2.5 kg/cow per d and becomes apparent after approximately 4 wk of exposure to extended light. Recent studies indicate that dry cows do not increase milk yield in the next lactation with only 21 d of exposure to SDPP, whereas a 35-d exposure to SDPP is sufficient to produce such a response in the next lactation (Reid et al., 2004; Velasco et al., 2006). Collectively, these studies support the concept that milk yield in cattle is greatly affected by varying the photoperiod exposure during the lactation cycle.
Substantial evidence has now been generated for a connection among photoperiod, PRL sensitivity, and shifts in mammary gland development and function in cattle. For example, dry cows exposed to LDPP have greater circulating PRL relative to SDPP but have reduced transcription of PRL receptor (PRL-R) mRNA in the mammary gland relative to SDPP cows (Auchtung et al., 2005; Wall et al., 2005a). Because postreceptor events are amplified and modulated within the cell, one can assume that greater relative receptor expression equates to greater sensitivity to hormonal stimulation. Although effects of photoperiodic variation on PRL-R expression at the protein level have not been confirmed in bovine mammary tissue, there is evidence that leukocytes from cows under SDPP exhibit greater sensitivity to PRL in vitro (Auchtung and Dahl, 2004). Thus, cattle on SDPP appear to have higher PRL sensitivity than cattle on LDPP.
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PROLACTIN SIGNALING IN RESPONSE TO PHOTOPERIOD
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At the cellular level, it was hypothesized that the effect of SDPP relative to LDPP in dry cows is due to increased growth of mammary epithelial cells in response to enhanced PRL signaling converging via effects on the IGF axis. To examine this, mammary biopsies were obtained from multiparous Holstein cows at –46, –24, –9, and +11 d relative to calving, and rates of [3H]-thymidine incorporation into DNA were quantified in vitro (Wall et al., 2005a). Abundance of IGF-I, IGF-II, and IGFBP-5 mRNA was assessed by quantitative real-time reverse transcription PCR. For both SDPP and LDPP treatments, cell proliferation rate increased significantly from –46 to –9 d and then decreased significantly during lactation. However, timing of the proliferative response differed between treatments, in which proliferation rate increased between –46 and –24 d in SDPP cows and was significantly greater than in LDPP cows at –24 d. In LDPP-treated cows, the increase in proliferation did not occur until –9 d.
Expression of IGF-II was significantly greater during late gestation in SDPP vs. LDPP cows, and IGFBP-5 mRNA during lactation was significantly greater in LDPP cows vs. SDPP cows. Expression of IGF-I did not differ over time or between treatments (Wall et al., 2005a). It was concluded that exposure to SDPP during the dry period elicits earlier mammary cell proliferation relative to LDPP. Despite the lack of treatment difference in IGF-I expression, the lower IGFBP-5 expression in SDPP cows coupled with increased IGF-II expression may enhance mammary cell growth and survival. Further, temporal differences between treatments suggest the existence of a critical window at approximately 20 d prepartum wherein photoperiod affects mammary gland development in dry cows.
Of additional interest is evidence that suppressors of cytokine signaling (SOCS) can modulate PRL signaling in mammary tissue. Lindeman et al. (2001) demonstrated that the defective mammary development in mice with hemideletion of the PRL-R gene could be rescued by additional deletion of a single SOCS-1 allele, and they suggested a role for SOCS-1 in suppressing lactation before parturition. Tam et al. (2001) found that during an established lactation in rats, pup removal increased mammary SOCS-3 expression, which may contribute to mammary gland insensitivity to PRL. Finally, Park et al. (2002) reported that mice in which the caveolin-1 (CAV1) gene had been deleted exhibited accelerated lobuloalveolar development, premature lactation, and hyperactivation of the Janus kinase-signal transducers and activators of transcription signaling pathways, indicating that this gene is a novel suppressor of cytokine signaling. These studies provide powerful evidence indicative of a critical role for SOCS in the modulation of PRL-induced mammary development. Indeed, there is now evidence in cattle of a relationship among PRL sensitivity, SOCS signaling, and milk production.
Wall et al. (2005b) hypothesized that altered expression of SOCS, as a result of different day length, modulates mammary sensitivity to circulating PRL during the dry period. Transcription of mRNA for SOCS-1, SOCS-2, and SOCS-3, cytokine-inducible SH2-containing protein (CIS) and interferon-
receptor was assessed by quantitative real-time reverse transcription PCR in mammary tissue collected sequentially during the dry period and early lactation (Wall et al., 2005b). Mammary expression of SOCS-2 and CIS was low during the dry period and then increased significantly in lactation. Significantly greater abundance of both CIS and SOCS-2 mRNA was observed in LDPP cows compared with SDPP cows at –24 d relative to calving. Expression of CIS was also higher at +11 d in LDPP cows compared with SDPP cows. Expression of SOCS-3 and interferon- receptor increased significantly from –46 to –9 d and remained high during lactation for both treatments. These results indicate that cows exposed to LDPP during the dry period experience higher SOCS expression, which may impair PRL-induced mammary growth and lead to inferior milk production in the subsequent lactation. The increase in mammary SOCS expression from pregnancy into lactation implies a functional role for SOCS in bovine mammary gland during lactogenesis. Furthermore, the temporal pattern of changes in SOCS-2 and CIS expression is consistent with the hypothesis that both are involved in regulating mammary proliferation, particularly around 20 d before calving.
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PROLACTIN SIGNALING AT OTHER TISSUES
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Previous studies on photoperiod manipulation clearly show that exposure to SDPP during the dry period substantially improves subsequent lactation performance in the order of 3 to 4 kg/d (Miller et al., 2000; Auchtung et al., 2005; Crawford et al., 2005; Velasco et al., 2006). Results of parallel studies provide evidence that the same SDPP treatment reduces circulating PRL but increases PRL-R mRNA expression in mammary tissues (Auchtung et al., 2003, 2004, 2005). The observed inverse relationship between PRL and PRL-R expression is also apparent in hepatocytes and leukocytes, providing evidence that the pathway is commonly adapted by multiple tissues related to support of milk production. Relative to leukocytes collected from cows on LDPP, those collected from SDPP cows are more responsive to PRL in vitro, providing functional evidence to support the concept of greater PRL sensitivity under SDPP treatment (Auchtung and Dahl, 2004). Finally, at least one study provides support for improved functional immune outcomes, namely lower somatic cell counts and reductions in mastitis during early lactation, in dry cows exposed to SDPP vs. LDPP (Auchtung et al., 2004). Overall, these studies strongly support the hypothesis that photoperiod-dependent alterations in PRL sensitivity mediate physiological changes in leukocytes that translate into differences in immune function.
Taking into account the hypothesis that environmentally induced shifts in PRL, and therefore PRL-R, alter liver lipid metabolism, Connor et al. (2007) investigated the effect of SDPP and LDPP exposure on hepatic gene expression of critical enzymes for lipid metabolism. Samples were collected from growing steers, as done by Auchtung et al. (2003), who established an inverse relationship between circulating PRL concentrations and PRL-R mRNA abundance. Briefly, liver biopsies were collected after 3 and 6 wk of exposure to a SDPP or LDPP. Relative to LDPP, the expression of acetyl-CoA carboxylase decreased in SDPP steers after 6 wk of treatment. This outcome is consistent with a lowered incidence of fatty liver, because Savage et al. (2006) showed that blocking acetyl-CoA carboxylase expression in rat liver increased hepatic fat oxidation, inhibited hepatic lipogenesis, and decreased hepatic concentration of malonyl-CoA. Exposure to SDPP (Connor et al., 2007) also decreased very long chain acyl-CoA dehydrogenase (ACADVL) expression in steers after 6 wk of treatment. This result might initially be interpreted as a negative outcome in terms of preventing fatty liver, because fatty liver is associated with increased hepatic de novo fatty acid synthesis and decreased β-oxidation. As ACADVL catalyzes the first step in β-oxidation of fatty acids, it appears that SDPP is reducing fatty acid oxidation in the liver. However, Loor et al. (2005) observed a positive association between ACADVL mRNA and elevated concentrations of hepatic triacylglycerol and circulating NEFA, which are hallmarks of fatty liver. Thus, a reduction in ACADVL activity would likely be reflected by lower circulating NEFA and lower triacylglycerol content of liver.
When compared with LDPP, treatment with SDPP decreased the expression of sterol regulatory binding transcription factor 1 (SREBP-1c), a transcription factor for lipid biosynthesis, in steers after 6 wk of exposure. Increased SREBP-1c expression in mice is associated with increased fatty acid synthesis and triglyceride accumulation in liver and may contribute to steatosis in diabetic mice (Shimomura et al., 1999). Therefore, a decrease in SREBP-1c expression should help to prevent fatty liver. Compared with LDPP, there was a tendency for an increase in fatty acid synthase expression in SDPP steers after 3 wk of treatment, but the effect disappeared by 6 wk. Collectively, these results support the hypothesis that exposure to SDPP should limit the development of fatty liver and that environmentally induced decreases in circulating PRL are associated with improvements in hepatic function during the transition into lactation. These results are also consistent with the concept that multiple tissues exhibit a coordinated response to PRL signaling in preparation for the next lactation.
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HEAT STRESS EFFECTS ON PROLACTIN AND LACTATION
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Underlying the production responses described previously, overwhelming evidence is available to indicate that photoperiod manipulation affects mammary and immune function in mature cows, both in vitro and in vivo (Auchtung et al., 2004, 2005). Mechanistically, shifts in PRL sensitivity appear to mediate the changes observed in mammary, immune, and hepatic function, where there is a notable inverse relationship between circulating PRL and the level of PRL-R expression in many tissues (Auchtung et al., 2003, 2004; Auchtung and Dahl, 2004; Wall et al., 2005a,b). Knowledge of the influence of another environmental cue that perturbs PRL release, namely heat stress, has led to the hypothesis that the negative effect of heat stress on cow performance is mediated through disturbed PRL signaling.
Following the overall hypothesis that PRL sensitivity during the transition into lactation is a critical factor in determining ultimate milk yield and periparturient health, a plausible mechanistic explanation for the observed effects of heat stress is a decrease in PRL sensitivity. For example, heat stress increases circulating PRL in cattle (Tucker et al., 1991). Further, heat stress has profound negative effects in lactating cows, and cows that experience heat stress during the dry period have persistently depressed performance in the subsequent lactation (Aharoni et al., 2000; Collier et al., 2006). Recent evidence indicates that cooling dry cows improves their subsequent production, although that data set was limited to the initial 60 d of lactation, and cooling was not implemented during the entire dry period (Urdaz et al., 2006). It is interesting to speculate that cows that are cooled during the dry period would be expected to have reduced circulating PRL but greater PRL-R expression relative to dry cows that are heat-stressed, but evidence to confirm that hypothesis is not currently available.
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CONCLUSIONS
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It is clear that photoperiod influences mammary and immune function during late gestation in dairy cows, and this effect has a persistent effect on the subsequent lactation. Further, physiological responses to PRL are critical to the photoperiodic response in dry cows and thus may be a factor in other environmental effects (e.g., temperature and stress). The short day response in dry cows is a sequential process that requires at least 40 d to complete, and that timeline is consistent with a series of cellular responses in PRL signaling pathways. These include the observation that IGF-II increases are associated with some of the mammary growth effects of short days and that SOCS pathways are suppressed under short days. Altered PRL signaling in other tissues (e.g., liver and immune) suggest a coordinated action to support the lactation response in dry cows subjected to SDPP. Extending these observations to another environmental factor (i.e., heat stress), it is reasonable to hypothesize that excessive PRL release under high temperatures is related to some of the deleterious effects of heat stress in dry cows. Knowledge of the crucial role of PRL signaling during the dry period may lead to improvements in dry cow management via recommendations on housing or other treatments to reversibly suppress disturbances in PRL secretion and improve PRL sensitivity during this critical stage of the lactation cycle.
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Footnotes
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1 The author thanks T. McFadden, E. Wall (Univ. Vermont), E. Connor (USDA-ARS, Beltsville, MD), T. Auchtung-Montgomery, E. Reid, H. Crawford, K. Karvetski, and J. Velasco (Univ. Illinois) for assistance in the conduct of the studies described herein. Funding for these efforts was provided in part by USDA-CSREES-NRI, US-Israel BARD, Deere and Co., Illinois C-FAR, and UF-IFAS. 
2 Presented at the Eighth International Workshop on the Biology of Lactation in Farm Animals held in Pirassununga, Brazil, August 21–23, 2006.
3 Corresponding author: gdahl{at}ufl.edu
Received for publication May 30, 2007.
Accepted for publication July 30, 2007.
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LITERATURE CITED
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Aharoni, Y., A. Brosh, and E. Ezra. 2000. Short Communication: Prepartum photoperiod effect on milk yield and composition in dairy cows. J. Dairy Sci. 83:2779–2781.[Abstract]
Auchtung, T. L., and G. E. Dahl. 2004. Prolactin mediates photoperiodic immune enhancement: Effects of administration of exogenous prolactin on circulating concentrations, receptor expression, and immune function in steers. Biol. Reprod. 71:1913–1918.[Abstract/Free Full Text]
Auchtung, T. L., P. E. Kendall, J. Salak-Johnson, T. B. McFadden, and G. E. Dahl. 2003. Photoperiod and bromocriptine treatment effects on expression of prolactin receptor mRNA in bovine liver, mammary gland and peripheral blood lymphocytes. J. Endocrinol. 179:347–356.[Abstract]
Auchtung, T. L., A. G. Rius, P. E. Kendall, T. B. McFadden, and G. E. Dahl. 2005. Effects of photoperiod during the dry period on prolactin, prolactin receptor, and milk production in dairy cows. J. Dairy Sci. 88:121–127.[Abstract/Free Full Text]
Auchtung, T. L., J. L. Salak-Johnson, D. E. Morin, C. C. Mallard, and G. E. Dahl. 2004. Effects of photoperiod during the dry period on cellular immune function of dairy cows. J. Dairy Sci. 87:3683–3689.[Abstract/Free Full Text]
Bourdon, R. M. 1997. Heritability of dairy traits. Page 164 in Understanding Animal Breeding. 2nd ed. Charles E. Stewart Jr., ed. Prentice Hall, Upper Saddle River, NJ.
Collier, R. J., G. E. Dahl, and M. J. Van Baale. 2006. Major advances associated with environmental effects on dairy cattle. J. Dairy Sci. 89:1244–1253.[Abstract/Free Full Text]
Connor, E. E., E. D. Thomas, and G. E. Dahl. 2007. Photoperiod alters metabolic gene expression in bovine liver potentially through suppressors of cytokine signaling. J. Anim. Sci. 85(Suppl. 1):208. (Abstr.)
Crawford, H. M., J. Dauderman, D. E. Morin, T. B. McFadden, and G. E. Dahl. 2005. Evidence of a role of prolactin in mediating photoperiodic effects during the dry period. J. Anim. Sci. 83(Suppl. 1):363. (Abstr.)
Dahl, G. E., B. A. Buchanan, and H. A. Tucker. 2000. Photoperiodic effects on dairy cattle: A review. J. Dairy Sci. 83:885–893.[Abstract]
Dahl, G. E., and D. Petitclerc. 2003. Management of photoperiod in the dairy herd for improved production and health. J. Anim. Sci. 81(Suppl. 3):11–17.[Abstract/Free Full Text]
Lindeman, G. J., S. Wittlin, H. Lada, M. J. Naylor, M. Santamaria, J.-G. Zhang, R. Starr, D. J. Hilton, W. S. Alexander, C. J. Ormandy, and J. Visvader. 2001. SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 15:1631–1636.[Abstract/Free Full Text]
Loor, J. J., H. M. Dann, R. E. Everts, R. Oliveira, C. A. Green, N. A. Guretzky, S. L. Rodriguez-Zas, H. A. Lewin, and J. K. Drackley. 2005. Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol. Genomics 23:217–226.[Abstract/Free Full Text]
Miller, A. R. E., R. A. Erdman, L. W. Douglass, and G. E. Dahl. 2000. Effects of photoperiodic manipulation during the dry period of dairy cows. J. Dairy Sci. 83:962–967.[Abstract]
Park, D. S., H. Lee, P. G. Frank, B. Razani, A. V. Nguyen, A. F. Parlow, R. G. Russell, J. Hulit, R. G. Pestell, and M. P. Lisanti. 2002. Caveolin-1 deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade. Mol. Biol. Cell 13:3416–3430.[Abstract/Free Full Text]
Reid, E. D., R. L. Wallace, T. B. McFadden, and G. E. Dahl. 2004. The effects of a 21 day short day photoperiod treatment during the dry period on dry matter intake and subsequent milk production in cows. J. Anim. Sci. 82(Suppl. 1):424. (Abstr.)
Savage, D. B., C. S. Choi, V. T. Samuel, Z. X. Liu, D. Zhang, A. Wang, X. M. Zhang, G. W. Cline, X. X. Yu, J. G. Geisler, S. Bhanot, B. P. Monia, and G. I. Shulman. 2006. Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2. J. Clin. Invest. 116:817–824.[CrossRef][Medline]
Shimomura, I., Y. Bashmakov, and J. D. Horton. 1999. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J. Biol. Chem. 274:30028–30032.[Abstract/Free Full Text]
Tam, S. P., P. Lau, J. Djiane, D. J. Hilton, and M. J. Waters. 2001. Tissue-specific induction of SOCS gene expression by PRL. Endocrinology 142:5015–5026.[Abstract/Free Full Text]
Tucker, H. A., L. T. Chapin, K. J. Lookingland, K. E. Moore, G. E. Dahl, and J. M. Evers. 1991. Temperature effects on serum prolactin concentrations and activity of dopaminergic neurons in the infundibulum/pituitary stalk of calves. Proc. Soc. Exp. Biol. Med. 197:74–76.[CrossRef][Medline]
Tucker, H. A., and R. K. Ringer. 1982. Controlled photoperiodic environments for food animals. Science 216:1381–1386.[Free Full Text]
Urdaz, J. H., M. W. Overton, D. A. Moore, and J. E. Santos. 2006. Technical note: Effects of adding shade and fans to a feedbunk sprinkler system for preparturient cows on health and performance. J. Dairy Sci. 89:2000–2006.[Abstract/Free Full Text]
Velasco, J. M., K. E. Karvetski, E. D. Reid, T. F. Gressley, R. L. Wallace, and G. E. Dahl. 2006. Short day photoperiod increases milk yield in cows with a reduced dry period length. J. Anim. Sci. 84(Suppl. 1):147. (Abstr.)
Wall, E. H., T. L. Auchtung, G. E. Dahl, S. E. Ellis, and T. B. McFadden. 2005a. Exposure to short day photoperiod enhances mammary growth during the dry period of dairy cows. J. Dairy Sci. 88:1994–2003.[Abstract/Free Full Text]
Wall, E. H., T. L. Auchtung-Montgomery, G. E. Dahl, and T. B. McFadden. 2005b. Short communication: Short day photoperiod during the dry period decreases expression of suppressors of cytokine signaling in the mammary gland of dairy cows. J. Dairy Sci. 88:3145–3148.[Abstract/Free Full Text]
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Abstract
INTRODUCTION
