Dietary manipulations to improve embryonic survival in cattle William Thatcher*, José E.P. Santos, Charles R. Staples

Transcription

1 Available online at Theriogenology 76 (2011) Advances in Bovine Reproduction and Embryo Technology Dietary manipulations to improve embryonic survival in cattle William Thatcher*, José E.P. Santos, Charles R. Staples Department of Animal Sciences, IFAS, University of Florida, Gainesville, Florida, USA Received 1 May 2011; received in revised form 21 May 2011; accepted 25 May Abstract High-producing dairy cows are subfertile. Hormonal and metabolic responses associated with homeorrhetic and homeostatic regulatory responses to partition nutrients for lactation, coupled with management, contribute to the reduction in fertility. Systems of reproductive management partially restore herd reproductive performance and provide a basis to access the impact of targeted nutritional strategies to further improve postpartum health and reproduction. Increasing the number of days feeding prepartum diets with a negative dietary cation-anion difference (DCAD), combined with adequate energy, protein, amino acids, and trace/macrominerals, improves the subsequent pregnancy rate. Likewise, supplementation of organic Se in the transition period and lactation improves immune function, uterine health, and subsequent reproductive performance under conditions of Se insufficiency. A basic understanding of the regulatory processes between nutrient partitioning and reproduction has led to the development of dietary strategies that benefit both lactation and reproduction. Postpartum increases in dietary nonstructural carbohydrates (i.e., glucogenic diets) increases ovarian activity in either intensive or extensive systems. Furthermore, sequential feeding of glucogenic-lipogenic diets enhances the proportion of cows pregnant by 120 d of lactation. Fatty acids of the n-6 and n-3 families act as nutraceuticals, altering innate immune responses and subsequent gene expression within the uterus to complement the sequential processes of follicle and embryo development and survival of the embryo and fetus. Selective or sequential feeding of lipogenic diets can benefit reproductive and immunological responses of lactating dairy cows and extensively managed beef cows Elsevier Inc. All rights reserved. Keywords: Cow; Fertility; Lactation; Neutrophils, Nutrition Contents 1. Introduction The biological window of transition Nutritional management to improve pregnancy per AI or pregnancy rate Mineral supplementation in transition diets Dietary cation-anion difference (DCAD) Dietary supplementation with organic trace minerals Dietary supplementation with amino acids and protein Sequential feeding of glucogenic-lipogenic diets Dietary inclusion of fat-enriched feedstuffs Selective feeding of lipogenic diets: endocrine, metabolic and tissue responses Selective or sequential feeding of lipogenic diets on reproductive and immunological responses * Corresponding author. Tel.: , Ext. 244; fax address: thatcher@ufl.edu (W. Thatcher) X/$ see front matter 2011 Elsevier Inc. All rights reserved. doi: /j.theriogenology

2 1620 W. Thatcher et al. / Theriogenology 76 (2011) Conclusion Acknowledgments References Introduction It has been well documented that pregnancy per insemination in dairy cattle has declined as milk production has increased. There has been considerable debate regarding whether genetic selection for milk production per se caused the decline in fertility, or whether management (e.g., nutrition, milking and reproductive manipulations, housing/cow comfort, abatement of environmental stresses, etc.) and health care have not been optimized to meet the physiological and metabolic needs of higher producing dairy cows to enable them to reproduce optimally [1]. Indeed, it is likely that these entire multiple and interactive components have contributed to reductions in fertility. Nevertheless, major advancements in understanding these basic and applied processes are beginning to overcome constraints to increased fertility, leading to increases in herd pregnancy rates. Many dairy herds with high producing cows managed in a variety of environments obtain satisfactory reproductive performance. The integration of genomics, bioinformatics, transcriptomics, proteomics, and metabolomics, as applied to the processes of reproduction, nutrition, milk production, growth, and pregnancy, are leading to major advancements in dairy production when integrated with holistic management systems. In lactating dairy cattle, systems of programmed timed insemination have been developed to achieve pregnancy per AI of 45% for first and second services [2,3]. These improvements have come from an advanced understanding of the processes of follicle and CL development, CL regression, ovulation, and the coordination of these processes in a physiological manner to meet the unique metabolic and endocrine challenges encountered in lactation. Such reproductive management programs (i.e., fertility programs that increase submission rates and pregnancies per AI) have further documented and substantiated the importance of the transition period and postpartum health as biological windows that are likely imprinting the degree of reproductive competence of cows entering the breeding period [4]. This review focuses on potential dietary and nutraceutical approaches which may improve reproductive performance of lactating dairy cows. 2. The biological window of transition As cows undergo transition from the pregnant nonlactating state to lactation, homeorrhesis orchestrates the mobilization of tissues, primarily adipose, to meet the needs to produce milk at a time when dietary nutrient intake is not sufficient to support lactation. Thus, the cow is in a period of declining nutrient status that may begin in the last 2 wk before calving, but becomes markedly negative early in the postpartum period, with a nadir between 10 and 15 d postpartum, and a return to a positive energy balance at approximately 5 to 8 wk of lactation. This dynamic period is reflected by changes in body condition (BCS) that approximates lipid stores associated with subcutaneous fat [5]. Pregnancy responses to BCS were characterized in four large commercial dairy farms in California [6]; cows with a BCS 3.00 (5-point scale) either at calving or at AI had marked reductions in pregnancy per AI at 30 and 58 d after insemination. Furthermore, the change in BCS from calving to AI also influenced fertility; cows that lost 1 BCS unit had lower pregnancy per AI and more than double the odds of pregnancy loss [6]. Based upon these data [6], pregnancy per AI was 38% when BCS was 3.0 at calving and at time of AI, and was not related to milk yield. If higher producing dairy cows are healthy and managed well, then satisfactory reproductive performance ensues [4]. Leblanc [7], after reviewing the literature, concluded that any association between milk yield and the probability and timing of pregnancy was not clear, either among cows with distinct production in a population at one time, or with increasing milk production over time. Roche et al [8] asserted that if lipid metabolism was regulated solely by homeostatic controls in early lactation, then increased caloric intake should in principle eliminate lipid mobilization. However, most attempts to reduce lipid mobilization by feeding energy-rich diets (e.g., nonstructural carbohydrates or supplemental fats) in the transition and/or early postpartum periods have, for the most part, been unsuccessful in reducing lipid mobilization, based upon plasma concentrations of nonesterified fatty acids (NEFAs). Nevertheless, this does not preclude that targeted changes in dietary composition may alter other biochemical and physiological aspects associated with disorders in mineral metabo-

3 W. Thatcher et al. / Theriogenology 76 (2011) lism (hypocalcemia and hypomagnesemia), immune system function (retained placenta, metritis, endometritis, and mastitis), and energy metabolism (ketosis, fatty liver, subacute ruminal acidosis, anovulation). Indeed, these various production disorders are interrelated and associated with poor reproductive responses [4]. Potential dietary management to minimize their occurrence warrants further investigation in order to target improvements in reproductive efficiency. A brief synopsis of the homeorrhetic changes occurring with the onset of lactation (i.e., periparturient period) sets the foundation to develop potential nutritional interventions during this critical period. With the decrease in dry matter intake (DMI) that begins approximately 10 d before parturition and insufficient caloric intake to meet the requirements in the first weeks of lactation, the lactating dairy cow mobilizes adipose tissue as NEFAs. Glucose that is synthesized from propionic acid through hepatic gluconeogenesis is readily utilized by the mammary gland for milk synthesis, resulting in hypoglycemia and hypoinsulenemia. Excessive -oxidation of fatty acids by the liver leads to ketogenesis and increases plasma concentrations of -hydroxybutyrate, symptomatic of sub-clinical and clinical ketosis. Furthermore, all these events tend to be exacerbated in overconditioned cows that have reduced appetite and are more prone to lipid mobilization. Metabolic hormones, such as growth hormone (GH), insulin, and insulin-like growth factor-1 (IGF-1) are associated casually with differential temporal changes in metabolites that reflect nutrient partitioning. Blood GH concentrations are usually elevated around calving, but are greater in cows selected for higher milk production [9]. High plasma GH concentrations stimulate lipolysis, thereby contributing to the rise in plasma NEFAs as energy balance declines. High concentrations of GH and NEFAs antagonize the action of insulin, thereby contributing to a state of insulin resistance in early postpartum cows. This coordinated metabolic and endocrine response alters post-absorptive carbohydrate metabolism, such that a net reduction in glucose uptake occurs in peripheral tissues (i.e., muscle and adipose), to favor mammary uptake. During this early period, GH is uncoupled from its receptors in the liver. As caloric intake increases, so do insulin concentrations. Increased plasma insulin concentrations, within the normal physiological range during periods of negative energy status, increase hepatic GH receptor 1A and IGF-I mrna, as well as plasma IGF-1 [10]. Both insulin and IGF-1 are important hormonal cues that control ovarian follicular activity, leading to ovulation and regular ovarian cycles. Acting on the liver and other organs, GH influences the secretion of IGF-1 and leptin, hormones that help regulate feed intake and might directly affect reproduction. Understanding the coordination and regulation of periparturient energy status with metabolic and reproductive hormones in high-producing dairy cows are essential to develop nutritional strategies to improve reproduction. The classical studies of Beam and Butler [11,12] documented that the first wave of ovarian follicular development in dairy cows begins 5 to 7 d postpartum, regardless of energy status. This early growth of follicles is caused by increased blood FSH concentrations. However, the functional outcome of the first-wave follicle varies depending upon the associated metabolic and hormonal changes of the cow. Approximately 40 to 50% of cows ovulate their first-wave dominant follicle, which has increased steroidogenic capacity in association with greater plasma concentrations of insulin and IGF-1. Subsequent studies [13] segregated cows according to their postpartum follicle dynamics as ovulatory (n 17), nonovulatory high estradiol (n 6), nonovulatory low estradiol (n 24), or cystic (n 8). Differences among cows in DMI and energy status in the various follicle categories were evident as early as 3 wk prepartum and 0 to 30 d postpartum, respectively. Ovulatory cows had greater pre-and postpartum DMI and energy status compared with nonovulatory low estradiol cows. Differences among groups in energy status were reflected in differences in postpartum concentrations of metabolic hormones and metabolites, with greater concentrations of insulin, IGF-1, and glucose, but lower NEFA concentrations in ovulatory cows. The predictable early postpartum rise in FSH ensures that the developing follicle is exposed to a complement of metabolic and reproductive hormones associated with positive energy status. This sets the functional destiny of the reproductive system (i.e., coordinated gonadotrophin secretion, ovarian cyclicity, and oviductal/uterine competence to support pregnancy). In fact, in vitro stimulated bovine granulosa cells with FSH develop receptors for insulin, IGF-1, and GH [14,15], and increases in insulin and IGF-1 are associated with early first ovulation in dairy cows [12,16]. 3. Nutritional management to improve pregnancy per AI or pregnancy rate 3.1. Mineral supplementation in transition diets Dietary cation-anion difference (DCAD) The onset of lactation causes a marked and acute decline in blood Ca concentrations. The reduction in

4 1622 W. Thatcher et al. / Theriogenology 76 (2011) DCAD to negative values during the last 3 wk prepartum (i.e., 100 to 150 meq/kg of DM) minimizes the rapid decline in blood Ca. DeGaris et al. [17] investigated the effect of increasing the impact of number of days feeding prepartum diets on subsequent reproductive performance and health of dairy cows in three herds. Diets were formulated to provide more than adequate calories, protein, amino acids, and trace minerals, along with a negative DCAD and adequate macromineral supply suitable for prevention of mineralrelated disorders. Increasing the number of days of feeding the prepartum diet improved pregnancy rates. For each additional day of feeding, the risk of pregnancy increased by 2.1% (P 0.060) and by 2.2% (P 0.001) in Herds 2 and 3, respectively, but reduced pregnancy by 2.7% (P 0.07) in Herd 1. Proportion of cows pregnant tended to differ by 6 wk (P 0.06) and 21 wk (P 0.08) after the starting date for insemination with increased time of feeding the prepartum diet (Fig. 1). Cation-anion difference of the diet fed postpartum is also important for dairy cow performance. As described previously, it is important that DMI increase in the early postpartum period to stimulate coupling of the somatotrophic axis via insulin for stimulation of reproductive processes. A meta-analysis was conducted to examine potential empirical relationships between DCAD (using the equation of Na K Cl) and responses of lactating dairy cows [18]. The database was developed from 12 studies that included a total of Fig. 1. Survival graph for calving to conception and cumulative pregnancy for cows exposed to the pre-calving transition diet for 10, 10 to 20 and 20 d. Figure adapted from [17]. 17 trials, 69 dietary treatments, and 230 cows. Conclusions were that DCAD affected performance of lactating dairy cows such that maximum milk yield and feed intake were reached when DCAD was 340 and 400 meq/kg of feed DM, respectively. Blood ph and HCO 3 concentrations increased with increased DCAD, indicating improved acid-base balance of lactating dairy cows. Such a beneficial effect on feed intake in the postpartum period would also benefit reproductive responses including pregnancy per AI, but these results have apparently not been documented Dietary supplementation with organic trace minerals Trace minerals are required for various vital biological processes, including reproduction. Trace minerals bound to organic molecules such as amino acids appeared to be more available to tissues for regulation of cellular functions [19]. Their potential effects on reproduction in lactating dairy cows may be important to the integration of nutritional-reproductive management systems. Under the conditions of a Se inadequacy during the heat stress season in Florida, USA, an organic source of Se as selenized yeast was compared with inorganic Se when fed in total mixed rations (0.33 mg of Se/kg of feed DM), beginning at 26 d prepartum, through a minimum of 81 d postpartum [19]. Feeding organic Se elevated plasma Se concentrations, improved neutrophil function around parturition, immunoresponsiveness in multiparous cows, and uterine health, and increased second-service pregnancy per AI. A replicated experiment conducted concurrently in California failed to detect effects of organic Se on immune function, uterine health, or pregnancy per AI, possibly because of an adequate concentration of Se in plasma of control cows. However supplemental feeding of organic Se increased milk yield in both studies [19,20]. Hackbart et al [21] evaluated the effects of organic trace mineral supplementation on reproductive measures in lactating dairy cows. At dry off, cows were supplemented with either inorganic trace minerals or a mixture of inorganic and organic forms. The organic trace mineral supplement provided 40, 26, 70, and 100% of supplemented Zn, Mn, Cu, and Co, respectively, to the nonlactating cows, and 22, 14, 40, and 100% of supplemented Zn, Mn, Cu, and Co, to the lactating cows. Supplementation of organic trace minerals increased milk production by 3 kg/d at the last week of the study and decreased mean milk fat by 0.24 percentage units and mean BCS from 3.02 to Supplementation with organic trace minerals did not affect tissue concentrations of trace min-

5 W. Thatcher et al. / Theriogenology 76 (2011) erals and reproductive responses in dairy cows. Whether supplying all of supplemental Zn, Mn, Cu, and Co in organic versus inorganic form may have improved reproductive responses cannot be assessed from the present study Dietary supplementation with amino acids and protein Attention to optimizing metabolizable protein (MP) in diets is critical for mammary gland and fetal development, milk protein yield, and immune and reproductive functions. The National Research Council (NRC, [22]) recommends that dairy cows (body weight of 680 kg and 90 d in milk) producing 35 to 45 kg/d of 3.5% fat-corrected milk be fed diets of 10.2 to 11.0% MP and 15.2 to 16% crude protein (DM basis). Feeding diets high in crude protein or inadequate in fermentable carbohydrates can result in inefficient protein utilization and excess absorption of rumen ammonia N. The absorbed ammonia is detoxified into urea by the liver at a further energy cost in cows which are already in a negative energy state in early lactation. An inverse relationship of high serum ( 19 mg/dl) or milk urea N concentrations and pregnancy per AI indicates a potential negative influence of overfeeding degradable protein or underfeeding fermentable carbohydrates on fertility [23]. Santos et al [24] concluded that excess protein intake and increased urea and ammonia in body fluids can be toxic to embryos via impairment in their development. These effects seemed to be associated with alterations in uterine ph and granulosa cell function. Garnsworthy and colleagues [25 27] conducted a series of experiments to evaluate dietary supplementation with protein/amino acids, starch and fats to manipulate metabolic and reproductive hormones in a manner to improve reproduction. In a 2 2 factorial arrangement of treatments evaluating diets containing low or high amounts of MP with either low or high proportions of leucine, dairy cows fed high MP/high leucine diets produced 2.0 kg/d more milk than the other three treatment groups [27]. Insulin concentrations in plasma were greater in the high MP/low leucine group. However, diet did not influence timing of ovulation, number of follicles, size of the ovulatory follicle, size of the developing CL, or plasma concentrations of progesterone and estradiol. Cows fed the high MP diets had greater plasma urea N concentrations ( mg/dl) and lost more body weight ( 0.11 vs kg/d). Authors concluded that altering metabolic hormones through manipulation of amino acid supply and balance is unlikely to have a significant effect on ovarian function. The latter effects on increased plasma urea N and body weight loss would likely be associated with reduced fertility through altered embryo development [24] and delayed ovulation [6]. Postpartum reproductive and metabolic responses were altered when multiparous Holstein cows were assigned at calving to one of four diets arranged in a 2 2 factorial, with two dietary concentrations of ruminally degradable protein, 11.1 or 15.7% of DM, and supplemental fat as Ca salts of palm fatty acids fed at 0 or 2.2% of dietary DM [28,29]. Cows fed excess ruminally degradable protein had less ovarian follicular development, delayed first postpartum luteal activity (25.2 vs 38.6 d), accumulated less luteal tissue, and had lower plasma progesterone concentrations. These changes were likely mediated by the greater body weight loss, plasma concentrations of NEFA and insulin which were higher and lower, respectively. In dairy cows fed a 15.7% degradable protein diet, supplementing Ca salts of long-chain fatty acids doubled the number of CL, reduced the interval to first rise in progesterone by 6 d, doubled the number of normal luteal phases, and restored the pattern of accumulated plasma progesterone concentrations similar to that induced by lower ruminally degradable protein diets. Accumulated percent of cows pregnant by 120 d postpartum was increased from 52.3 to 86.4% by supplemental fat. Therefore, in cows fed excess ruminally degradable protein, feeding Ca salts of long-chain fatty acids diminished adverse changes in body weight, attenuation in ovarian activity, and pregnancy rate. Collectively, it is fairly clear that feeding diets excessive in crude protein with high rumen degradability should be avoided. Dietary proteins and essential amino acids do not appear to be driving complementary profiles of metabolic and reproductive hormones conducive to stimulating reproductive responses Sequential feeding of glucogenic-lipogenic diets Feeding a high-starch postpartum diet enhances circulating insulin concentrations, stimulates follicle development, and increases incidence of ovulations prior to 50 d of lactation [25,30]. However, high starch diets can suppress appetite by inducing satiety and shorter meals. Furthermore, diets designed to increase plasma insulin concentration had negative effects on blastocyst development following in vitro maturation and fertilization [24,31]. In contrast, a high-fat diet (5.9% DM) increased blastocyst formation compared with a moderate-fat diet (4.1% DM) [32], and lowered plasma insulin concentrations [26]. When cows were fed fat

6 1624 W. Thatcher et al. / Theriogenology 76 (2011) sources differing in fatty acid profile, no differences in blastocyst development were detected [33]. Consequently, fat feeding may benefit embryo development and also decrease insulin secretion. These results led to an initial feeding of a glucogenic diet until first ovulation to stimulate both follicle development and onset of ovarian cycles, followed by feeding a lipogenic diet during the breeding period to attenuate insulin secretion and increase fatty acid availability [34]. The concept was that the glucogenic diet would increase blood glucose and insulin, thereby promoting follicle development and restoration of postpartum ovulation. Conversely, the lipogenic diet during breeding would reduce insulin concentrations and improve oocyte competence and embryo quality. Indeed, plasma insulin concentration was elevated in cows fed the glucogenic diet, but dietary treatments did not affect the days to first rise in plasma progesterone, first estrus, or first AI. However, at 120 d postpartum, a greater proportion of cows fed the glucogenic-lipogenic diet combination were pregnant (60%; 9/15) compared with the other three treatments combined (27%; 12/45). Pregnancy response to sequential feeding is exciting and brings to bear several potential implications and/or questions. Perhaps the interpretation is that the proportion of pregnant cows by 120 d postpartum was normal for cows fed the glucogenic-lipogenic dietary sequence, but suppressed in the other diets. The concept that an early postpartum glucogenic diet may influence subsequent response to fat feeding can be extended to the specific class of fatty acids that are predominant in the fat. The recent report that linolenic acid added during in vitro maturation of bovine cumulus-oocyte complexes increased subsequent in vitro fertilization and blastocyst development [35] would support a biological response that may potentially benefit pregnancy per AI under field conditions with targeted feeding of lipids enriched in omega-3 fatty acids. A pasture-based experiment in New Zealand [36] clearly documented that feeding a greater amount of nonstructural carbohydrate (38.1 vs 17.8% of dietary DM) in the postpartum period reduced the occurrence of anovulatory cows (Fig. 2) and contributed to a greater percentage of pregnant cows by 6 wk of the breeding season (93 vs 76%). A further increase in fertility caused by a dietary reduction in glucogenic hormones (e.g., insulin) during the breeding period was not tested in the aforementioned experiment. In fact, first service pregnancy per AI did not differ between treatments. However, the earlier or greater occurrences of estruses and ovulations before the commencement of Fig. 2. Effect of postpartum diet on the proportion of anovulatory dairy cows. Diets were isocaloric (179 MJ of ME/cow/d), but differed in the ratio of structural to nonstructural carbohydrate contents. Diets consisted of pasture and pasture silage (PostP) or pasture and pasture silage supplemented with 5 kg of dry matter/cow/d of a barley and corn grain concentrate (PostC). Figure adapted from [36]. the breeding season may have benefited the accumulated occurrence of pregnancies [37] Dietary inclusion of fat-enriched feedstuffs Dietary supplementation with lipids was historically considered as an option to improve the energy status of the cow because of the increased caloric density of the diet. However, high fat diets in the first weeks of lactation can suppress DMI and elevate plasma NEFA concentrations, which might antagonize fertility. In an extensive review, Staples et al [38] concluded that fat feeding does not necessarily increase energy status of early lactation cows, although, in many cases, it benefited reproduction. Variable reproductive responses to fat feeding may be caused by the variation in the fatty acid profile of the supplemental fat and their availability to tissues. The long chain polyunsaturated fatty acids linoleic (C18:2 n-6) and -linolenic (C18:3 n-3) acids are considered essential to mammalian cells. An overview of long chain fatty acid effects on reproduction in cattle [39] identified potential reproductive windows that may influence fertility and addressed various issues that may account for variability in responses. Fat feeding stimulates follicle growth in cattle, but may be influenced preferentially by type of fat (e.g., polyunsaturated fatty acids). A major issue is to determine whether responsive follicles are optimal for subsequent embryo development and CL function. Increased progesterone concentrations in cattle in response to increased intake of fatty acids may represent increased steri-

7 W. Thatcher et al. / Theriogenology 76 (2011) odogenesis and/or reduced clearance. Various studies indicated that dietary supplementation with unsaturated fatty acids may improve embryo quality and development [24,39]. This has been further substantiated by in vitro studies [35]. Incorporation of -linolenic acid to the oocyte maturation medium increased cumulus cell expansion and development of oocytes to methapase II. These stimulatory effects increased subsequent cleavage rates, enhanced blastocyst development, and increased number of cells in both the inner cell mass and trophectoderm. In contrast, linoleic acid supplementation to bovine oocytes during maturation inhibited subsequent early embryo development [40]. These findings have profound implications regarding the type of fat supplement, and whether it has the appropriate composition of polyunsaturated fatty acids to potentially enhance oocyte maturation and embryo development. Long chain unsaturated fatty acids can influence cellular responses by interacting with intracellular ligands. Peroxisome proliferator-activated receptors (PPAR) are a family of nuclear receptors activated by selected long chain fatty acids, eicosanoids, and peroxisome proliferators. Three PPAR isoforms, encoded by separate genes, have been identified as PPAR, PPAR and PPAR, which upon ligand binding can affect transcription of target genes. The PPAR is expressed in the endometrium of lactating dairy cows on day 17 of the cycle (Fig. 3). Furthermore, PPAR functions in the response of bovine endometrial cells in culture to GH Fig. 3. Peroxissome proliferator activated-receptor antibody reactivity (brown nuclear staining) from a representative endometrial tissue section of a lactating dairy cow at Day 17 of the estrous cycle. Inset indicates reactivity when rabbit IgG is substituted for primary antibody. LE, luminal epithelium; S, subepithelial stroma; GE, glandular epithelium. Bar 100 m. and long chain omega-3 fatty acids (eicosapentaenoic acid; C20:5 [41]) Selective feeding of lipogenic diets: endocrine, metabolic and tissue responses Caldari-Torres et al [42] examined metabolic, endocrine and productive responses in cows fed fats rich in either saturated or linoleic fatty acids. Dietary treatments were from 28 d before to 49 d after calving. Feeding the supplement rich in linoleic acid increased plasma concentrations of IGF-1 and glucose, increased accumulated progesterone concentration in plasma, as well as a numerical elevation in insulin compared with cows fed the saturated fatty acid supplement. Such a postpartum pattern would be conducive to stimulate ovarian follicular development. Nevertheless, the supplement rich in linoleic acid caused milk fat depression. The energy sparing effect caused by the reduced milk fat synthesis resulted in a numerically greater mean postpartum energy balance for cows fed the high linoleic acid diet (4.7 vs 2.1 Mcal/d). The implication of these findings was that a decrease in energy utilization for milk permitted an earlier re-occurrence of luteal activity. An extensive study was designed to examine the potential effects of fat supplementation as Ca salts of fish oil on hormonal and endometrial gene expression associated with bovine GH (bst) treatments and pregnancy status (cyclic vs pregnant) at day 17 after a synchronized estrus [43,44]. Feeding of the dietary fish oil supplement began 10 d postpartum, compared with an isocaloric control ration containing whole cottonseed. Cows fed the fish oil supplement produced more milk and had lower plasma insulin concentrations through 53 d postpartum. Injections of 500 mg of bst 0 and 11 d after induced ovulation resulted in a clear sustained rise in plasma IGF-1, but the rise was appreciably less in cyclic cows fed fish oil (Fig. 4). Plasma insulin concentrations decreased in cyclic cows fed fish oil treated or not with bst compared with cyclic cows fed the control diet, with or without bst treatment (1.0 and 0.9 ng/ml vs 1.2 and 1.5 ng/ml, respectively). Clearly, dietary fat supplementation deceased both insulin and bst responsiveness of the liver to produce IGF-1 [43]. A major finding was that feeding fish oil supplement induced changes in endometrial gene expression somewhat similar to those induced by pregnancy [44]. Cyclic cows fed fish oil (without bst) had reduced moderate to heavy staining intensity for estrogen receptor- (ER ) in the luminal epithelium. Furthermore, within the superficial glandular epithelium, fish oil increased the amount of moderate and heavy

8 1626 W. Thatcher et al. / Theriogenology 76 (2011) Fig. 4. Profiles of plasma insulin-like growth factor 1 (IGF-1) concentrations of cyclic cows fed control diet no bovine somatotropin (bst) injections ( ), cyclic cows fed control diet with bst injections ( ), cyclic cows fed fish oil (FO) no bst injections ( ), cyclic cows fed FO with bst injections ( ), pregnant cows fed control diet no bst injections ( ), and pregnant cows fed control diet with bst injections ( )fromd0to17ofasynchronized estrous cycle. All three groups of cows given bst had greater (P 0.01) mean IGF-1 concentrations than cows not injected with bst. The FO-fed cows injected with bst had lower (P 0.05) IGF-I plasma concentrations and pregnant cows injected with bst had greater (P 0.05) plasma concentrations of IGF-1 than cyclic cows fed the control diet with bst injections as detected by homogeneity of regression. Pooled SEM for bst-treated and non-bst-treated cows were 22.2 and 29.9, respectively. Figure adapted from [43]. growth factor-1 stimulates early embryo development to the blastocyst stage [49,50]. Farin et al [51] indicated a more direct role for IGF-2, either of maternal or conceptus origin, in regulating the development of the ruminant conceptus, particularly during trophoblast elongation and maternal recognition. These findings suggest a temporal pattern of embryo development influenced first by IGF-1 to the blastocyst stage, with a transition to IGF-2 in the post-blastocyst stage with filamentous development of the conceptus and maintenance of the CL. This is somewhat supported by regulation of IGF-1 and IGF-2 mrnas in response to fish oil supplementation and bst (Fig. 5). Expression of IGF-1 decreased in the endometrium of pregnant cows compared with cyclic cows, and bst failed to stimulate IGF-1 mrna. Furthermore feeding cyclic cows fish oil reduced IGF-1 mrna (Fig. 5A). In contrast, expression of IGF-2 mrna increased in pregnant cows com- staining for the nuclear progesterone receptor. The cyclic cows fed fish oil (with or without bst injections) had reduced staining intensity of prostaglandin endoperoxide synthase-2 (PGHS-2) protein in the luminal epithelium compared with cyclic control cows (with or without bst). In pregnant cows fed the control diet, decreased abundancies of both ER mrna and protein, and oxytocin receptor (OTR) mrna were detected compared with no bst-cyclic cows [44]. These decreases are caused by bovine interferon-tau secreted by the conceptus and are considered essential to reduce pulses of PGF 2 secretion. This is particularly important, as PGHS-2 protein, localized to the luminal epithelium, was increased in pregnant cows. Cyclic cows fed fish oil with or without bst had reduced staining intensity of PGHS-2 protein in the luminal epithelium that would complement the anti-luteolytic processes of pregnancy. Slightly attenuated increases in IGF-1 following bst treatment in fish oil-fed cows [43] may partially explain the increase in pregnancy per AI achievable with bst treatment [45 47]. Furthermore, the hypoinsulinemic effect of fish oil might benefit embryo development to the blastocyst stage [24,48]. Insulin-like Fig. 5. Least squares means and SEM for uterine endometrial IGF 1 and IGF 2 mrna abundance (Panels A and B, respectively) at day 17 after a synchronized estrus (day 0) in lactating cyclic cows fed a control (Con) or fish oil-enriched lipid (FO) diets and pregnant (Preg) cows fed a control diet. Cows were injected with or without bst on days 0 and 11. x z Within each panel, means without a common superscript differed (P 0.05). Figure adapted from [43].

9 W. Thatcher et al. / Theriogenology 76 (2011) pared with day 17 cyclic cows and bst stimulated IGF-2 mrna in both cyclic and pregnant cows. Feeding cyclic cows fish oil elevated mrna comparable to that in pregnant cows, but expression was not further increased by bst treatment (Fig. 5B). These findings indicate a co-regulation of IGF-1 and IGF-2 by feeding FO to cyclic cows that favors their differential gene expression, as observed in pregnant cows at day 17. Such findings support the potential beneficial effects of FO and bst to support pregnancy. The coordination of nutraceutical and pharmacological treatments and responses, such as these with a dietary supplement enriched in fish oil fatty acids and bst, are likely to be synergistic. In spite of the large extent of ruminal biohydrogenation of dietary polyunsaturated fatty acids, their concentrations increase in tissues following dietary supplementation [52]. The critical question is what concentrations of specific fatty acids are needed in bovine tissues to alter cellular mechanisms, such as binding to PPAR receptors (Fig. 3) that alter gene transcription and protein synthesis. Polyunsaturated fatty acids can alter lactation, reproductive and immunological processes that may benefit animal productivity and fertility [53 57] Selective or sequential feeding of lipogenic diets on reproductive and immunological responses In addition to the type of supplemental fats containing various polyunsaturated fatty acids, it is important to delineate what reproductive windows may be altered to improve overall herd reproductive performance. Silvestre et al [53,54] designed an experiment to evaluate the effects of differential timing of supplementation of different Ca salts of fatty acids on reproduction, production and measures of innate immune responses. Holstein cows (n 1,380) were assigned randomly to be fed either transition diets supplemented with 1.5% of the DM as Ca salts of either palm oil (PO; mostly saturated and monounsaturated fatty acids) or safflower oil (SO; mostly linoleic acid). At 31 d postpartum, cows within each transition diet were randomized to receive either PO or Ca salts containing fish oil (FO) until 160 d postpartum. Transition and breeding diets did not affect the proportion of cows cycling at 74 d postpartum (80.0%). Overall first service pregnancy per AI at 30 and 60 d after insemination were 39.3 and 33.3%, respectively, and there were no effects of diets (Table 1). However, fewer cows fed FO diets lost their pregnancy (6.3 vs 13.6%). Perhaps the anti-inflammatory effects of the omega-3 FA in FO [54] fed during the breeding period reduced pregnancy losses. Furthermore, cows fed FO had an increased pregnancy per AI to the second service at both 30 d (36.2 vs 27.2%) and 60 d (34.5 vs 23.7%) after insemination. At second service, cows fed SO in the transition period followed by FO during the breeding period had the highest proportion pregnant (Table 1). The combination of reduced pregnancy loss at first AI and increased pregnancy at the second AI resulted in a greater proportion of cows fed FO pregnant after the first two postpartum inseminations. The mechanisms regarding the positive effects of FO supplementation are speculative, but could be associated with an improved ratio of IGF-2 to IGF-1 gene expression, and/or anti-inflammatory effects within the endometrium that are complementary to the immunosuppressive and antiluteolytic effects of the conceptus that maintain pregnancy. Table 1 Pregnancy per AI at the first and second inseminations evaluated at 32 and 60 d after AI and pregnancy loss of dairy cows fed fat supplements in four sequences. Diets* Diet contrasts (P value) PO-PO SO-PO PO-FO SO-FO C1 C2 C3 1 st AI D32 % (n) 38.7 (107/276) 35.8 (96/268) 39.1 (103/263) 35.8 (89/248) NS NS NS D60 % (n) 33.7 (92/273) 29.7 (79/266) 37.0 (97/262) 32.8 (81/247) NS NS NS Loss % (n) 11.5 (12/104) 15.9 (15/94) 4.9 (5/102) 7.9 (7/88) NS 0.05 NS 2 nd AI D32 % (n) 27.7 (43/155) 26.7 (41/154) 30.3 (44/154) 43.3 (65/150) NS D60 % (n) 21.0 (38/152) 22.5 (34/151) 27.3 (39/143) 41.3 (62/150) NS Loss % (n) 5.0 (2/40) 10.0 (4/38) 7.1 (3/42) 4.6 (3/65) NS NS NS * PO, Ca salts of palm oil; SO, Ca salts of safflower oil; FO, Ca salts containing fish oil. Contrasts were C1 (transition diets [PO-PO PO-FO vs SO-PO SO-FO]); C2 (breeding diets [PO-PO SO-PO vs PO-FO SO-FO]); and C3 (interaction of diets [PO-PO SO-FO vs PO-FO SO-PO]). NS, not significant.

10 1628 W. Thatcher et al. / Theriogenology 76 (2011) In the same series of experiments, feeding SO during the transition period improved early postpartum neutrophil bactericidal function, abundance of the L-selectin adhesion molecule, and neutrophil production of pro-inflammatory cytokines [54]. The SO dietary supplement elevated plasma concentrations of haptoglobin and fibrinogen. Conversely, feeding FO during the breeding period attenuated cytokine secretion from neutrophils. Collectively, strategic supplementation of fatty acids benefitted immune function early postpartum and exerted immunosuppressive effects during the breeding period, which could explain some of the improvement observed in fertility [53]. Juchem et al [55] evaluated the effect of feeding Ca salts of PO or of a blend of linoleic and trans-octadecenoic fatty acids to pre- and postpartum dairy cows. Lactating cows fed the blend of unsaturated FA were 1.5 times more likely to be pregnant at 27 or 41 d after AI, compared with cows fed PO. Improvements in pregnancy when cows were fed Ca salts of linoleic and trans-octadecenoic fatty acid blend were supported by increased fertilization and embryo quality measured at 5 d after a programmed timed AI [56]. An additional class of polyunsaturated fatty acids, the conjugated linoleic acids, has been evaluated for its effects on pregnancy of dairy cows. Feeding approximately 10 g/d of ruminally protected C18:2 trans-10, cis-12 improved fertility of dairy cows, although the probability of pregnancy was decreased beyond this amount [57]. Using survival analysis with an optimal intake of 8.0 g/d of C18:2 trans-10, cis-12, the median interval to first ovulation was less for cows fed the conjugated linoleic acid supplement than control cows (27 vs 37 d). Perhaps the earlier time of ovulation contributes to a higher probability of getting cows pregnant earlier in lactation. Castaneda-Gutierrez et al [58] detected greater plasma IGF-1 concentrations in dairy cows supplemented with conjugated linoleic acids in early lactation. This could contribute to stimulation in follicle development and enhancement in subsequent pregnancy rates. Lopes et al [59] conducted four insightful experiments to evaluate the effects of supplementation with Ca salts containing 40% linoleic and 2.7% linolenic acids on reproductive function of Bos indicus Nellore suckled beef cows. Across experiments, cows were supplemented on pasture with 0.1 kg/d of Ca salts, whereas control cows were supplemented with kaolin (Experiments 1 to 3) or mostly saturated and monounsaturated fatty acids (Ca salts of PO) during specific periods of reproductive management (Table 2). In all four experiments, supplementation with Ca salts of polyunsaturated fatty acids improved pregnancy per AI (Experiments 1, 2, and 4), or after timed embryo transfer (Table 2). These results substantiate that Table 2 Pregnancy per timed AI or timed embryo transfer of Bos indicus suckled cows supplemented or not with Ca salts of polyunsaturated fatty acids (40% linoleic and 2.7% linolenic acids) or either kaolim* or Ca salts of palm oil [adapted from 59]. Experiment Feeding relative to day of induced ovulation Pregnancy per AI or embryo transfer, % P value I (timed AI) Control D 11 to D (182/459) 0.04 Unsaturated fatty acids D 11 to D (231/451) II (timed AI) Control D 11 to D (82/262) a 0.05 Unsaturated fatty acids D 11 to D (108/302) a Unsaturated fatty acids D 11 to D (109/254) b III (embryo transfer) Control D0 to D (78/207) 0.07 Unsaturated fatty acids D0 to D (113/228) IV (timed AI) Saturated fatty acids D0 to D (85/239) 0.02 Unsaturated fatty acids D0 to D (127/265) a,b Values with different superscripts differ (P 0.05). * Aluminum silicate (rumen-inert indigestible substance). Cows offered 0.4 kg/d of a protein-mineral mix, in addition to 0.1 kg/d of Ca salts of polyunsaturated fatty acids (PF) or kaolin (control), from the beginning of synchronization protocol (d 11) to 28 d after timed AI (day 28). Control cows offered 0.4 kg/d of a protein-mineral mix, in addition to 0.1 kg/d of kaolin from the beginning of synchronization protocol (d 11) to 28 d after timed AI (day 28); PF28 cows offered PF from the beginning of synchronization protocol (d 11) to 28 d after timed AI (d 28); PF16 cows offered PF from d 11 to 16 and control from days 17 to 28 relative to timed-ai. Cows offered PF or control from the end of synchronization protocol (day 0) until day 28 (21 d after embryo transfer). Cows offered PF or 0.4 kg/d of a protein-mineral mix, in addition to 0.1 kg/d of Ca salts of palm oil for 28 d beginning after AI.

11 W. Thatcher et al. / Theriogenology 76 (2011) supplementing 0.1 kg/d of rumen-inert polyunsaturated fatty acids to beef cows, particularly after breeding, is a nutritional strategy to enhance reproductive performance. The mechanisms of improved pregnancy through supplemental feeding of linoleic and linolenic acids during this specific period are unknown. Possibilities include changes in luteal function or alterations in the metabolic hormones insulin, IGF-1 and IGF-2 that would favor later stages of embryo development. Supplying the correct amounts of omega-6 and omega-3 fatty acids for post-absorption by the gastrointestinal tract appears to be a key issue. Two recent studies using large numbers of dairy cows were conducted with supplementation rolled [60] or extruded flaxseed [61] that is rich in linolenic acid. Authors reported no benefit to fertility. Approximation of calculated differences in milk uptake of linolenic acid per day between flaxseed and no flaxseed diets differed by only 2.3 g/d [60] and 6.3 g/d [61], respectively. Whether these differences reflect sufficient availability of regulatory fatty acids to the reproductive tissues to achieve reproductive benefits needs to be examined. Fuentes et al [61] detected subtle decreases in PGFM concentrations in plasma in cows fed flaxseed, although progesterone concentrations were not altered. 4. Conclusion It is clear that the reproduction of the lactating dairy cow suffers when nutrient intake is inadequate. Selection for increased production partitions more nutrients to the mammary gland at the expense of body reserves. The state of negative nutrient balance in early lactation uncouples the somatotrophic axis, which influences follicle development, steroidogenesis and, eventually, delays first postpartum ovulation. Strategies have been proposed to overcome such shortage of nutrients. Feeding diets that promote glucose synthesis and increase blood insulin concentrations favor earlier resumption of the first postpartum ovulation. Similarly, supplementing diets of dairy cows with unsaturated fatty acids that suppress milk fat synthesis reduces the energetic cost for milk production and favors earlier ovulation and, potentially, pregnancy. Manipulating the prepartum diet to improve Ca homeostasis postpartum and assuring adequate length of exposure to this diet seems to be critical to postpartum fertility. During the transition and breeding periods, incorporating sequential feeding of glucogenic-lipogenic diets, respectively, or manipulating the fatty acid profile of the supplemental fat, such that it targets specific biological windows critical to reproduction, seem to be promising strategies to improve fertility of dairy cows. The beneficial effects to fertility of feeding polyunsaturated fatty acids might be enhanced when utilized in combination with hormonal treatments that stimulate embryo development, e.g., use of bovine GH. Acknowledgments This IETS Preconference Symposium Presentation by W.W. Thatcher and co-authors is dedicated to the memory of Dr. Dirk Zaaijer and his efforts to optimize health and reproductive performance of lactating dairy cows through nutritional management. Future Fertility Systems, Oude Holterweg 2, 7245 TM Laren (Gld), the Netherlands. References [1] Walsh SW, Williams EJ, Evans ACO. A review of the causes of poor fertility in high milk producing dairy cows. Anim Reprod Sci 2011;123: [2] Bisinotto RS, Ribeiro ES, Martins LT, Marsola RS, Greco LF, Favoreto MG, Risco CA, Thatcher WW, Santos JEP. Effect of interval between induction of ovulation and AI and supplemental progesterone for resynchronization on fertility of dairy cows subjected toa5dtimed AI program. J Dairy Sci 2010;93: [3] Thompson IM, Cerri RLA, Kim IH, Green JA, Santos JEP, Thatcher WW. Effects of resynchronization programs on pregnancy per artificial insemination, progesterone and pregnancyassociated glycoproteins in plasma of lactating dairy cows. J Dairy Sci 2010;93: [4] Santos JEP, Bisinotto RS, Ribeiro ES, Lima FS, Greco LF, Staples CR, Thatcher WW. Applying nutrition and physiology to improve reproduction in dairy cattle. In: Lucy MC, Pate JL, Smith MF Spencer TE, editors. Reproduction in Domestic Ruminants VII, Nottingham University Press, 2010, pp [5] Chagas LM, Bass JJ, Blache D, Burke CR, Kay JK, Lindsay DR, Lucy MC, Martin GB, Meier S, Rhodes FM, Roche JR, Thatcher WW, Webb R. Invited review: New perspectives on the roles of nutrition and metabolic priorities in the subfertility of high-producing dairy cows. J Dairy Sci 2007;90: [6] Santos JEP, Rutigliano HM, Sá Filho MF. Risk factors for resumption of postpartum cyclicity and embryonic survival in lactating dairy cows. Anim Reprod Sci 2009;110: [7] LeBlanc S. Assessing the association of the level of milk production with reproductive performance in dairy cattle. J Reprod Dev 2010;56:S1 S7. [8] Roche JR, Friggens NC, Kay JK, Fisher MW, Stafford KJ, Perry DP. Invited review: Body condition score and its association with dairy cow productivity, health, and welfare. J Dairy Sci 2009;92: [9] Weber WJ, Wallace CR, Hansen LB, Chester-Jones H, Crooker BA. Effects of genetic selection for milk yield on somatotropin, insulin growth factor-i, and placental lactogen in Holstein cows. J Dairy Sci 2007;90: [10] Butler ST, Marr AL, Pelton SH, Radcliff RP, Lucy MC. Insulin restores GH responsiveness during lactation-induced negative