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Theriogenology 69 (2008) 8897

Nutritional management of the donor cow

J.E.P. Santos a,*, R.L.A. Cerri a, R. Sartori b

School of Veterinary Medicine, University of California Davis, Tulare, CA 93274 USA b Embrapa Genetic Resources and Biotechnology, Brasilia, DF 70770-900, Brazil

Abstract Nutrition of the donor cow can inuence oocyte and embryo quality, which can affect the success of embryo transfer. Severe undernutrition compromised ovarian follicular development, with implications for superovulatory response and embryo quality. In postpartum lactating cows, undernutrition or inability to consume sufcient nutrients delayed resumption of ovulation, reduced the number of follicles, and compromised oocyte quality. Moderate undernutrition of nonlactating cows was unlikely to affect embryo quality; conversely, nonlactating animals on maintenance diets usually had better superovulatory responses and improved oocyte competence and embryo quality. The negative effects of overfeeding are thought to be mediated by alterations in endocrine cues, such as hyperinsulinemia and increased glucose and IGF-I, which may interfere with glucose transport in the embryo and increase apoptosis. Manipulating energy sources such as carbohydrates and fatty acids (FA) may inuence embryo viability, but the effects of FA were not consistent in vitro; increasing concentrations of unsaturated FA in follicular and embryonic cells usually improved embryo viability and resistance to cryopreservation. Excess protein intake and increased urea and ammonia in body uids can be toxic to embryos, impairing their development; these effects seemed to be associated with alterations in uterine pH and granulosa cell function. Likewise, toxins in feeds (e.g. gossypol), reduced embryo development and increased pregnancy losses. Diet of donor cows should be formulated to optimize the supply of nutrients to meet needs; however, manipulating energy intake, source of FA and protein content of donor diets, particularly moderate underfeeding in nonlactating cows, may further optimize responses. # 2007 Elsevier Inc. All rights reserved.
Keywords: Cow; Embryo; Lipid; Nutrition; Protein

1. Introduction The ultimate goal of bovine embryo transfer (ET) is to obtain oocytes and embryos of high quality from superior dams that will result in the birth of healthy calves. Success of these programs depends upon superovulatory responses, and oocyte and embryo quality. The increased use of ovum pick-up and IVF has created additional impetus for improved oocyte quality. Nutrition has a major impact on fertility of

* Corresponding author. Present address: Department of Animal Science, University of Florida, Gainesville. E-mail address: (J.E.P. Santos). 0093-691X/$ see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.09.010

cattle, but less is known about the effects of nutritional status and the role of specic nutrients on basic aspects of superovulatory response, oocyte and early embryo quality and pregnancy following ET. Many physiological factors inuence the number of follicles recruited into a follicular wave, the competence of the oocytes within those follicles, and the ability of fertilized ova to result in embryos of high developmental potential. Nutritional manipulation can alter the metabolic and endocrine milieu to which oocytes and embryos are subjected; events occurring early in development, from a primary follicle to fertilization, may determine future fertility. A major impediment to understanding the inuences of nutrition of the donor cow is that most data were

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derived from studies using in vitro systems; they provided interesting ideas and allowed for mechanistic understanding of the cellular and molecular impacts of nutrients on the oocyte and embryo. However, the inability to mimic in vivo responses cannot be ignored when interpreting these results. Although alterations in culture media and conditions can improve embryo development in vitro [1,2], typically not more than 30% blastocysts are produced in IVF, whereas more than 60% blastocysts may be collected from cows on Days 6 and 7 after AI. It is clear that some components of the diet can impinge upon oocyte and embryo viability, and attempts to alter the supply of nutrients can result in improvements in ET. 2. The postpartum dairy cow and energy balance impacts donor performance The postpartum period is characterized by alterations in follicle development and hormone proles because of the combined inuences of the previous pregnancy and the impact of lactation. Delayed postpartum cyclicity in beef cows may compromise fertilization [3]; Breuel et al. [4] reported fertilization rates of only 75% in lactating beef cows, whereas fertilization rates averaged 98.6% in nonlactating beef cows [3]. In lactating dairy cows, genetic selection for increased milk yield altered physiological aspects of reproduction in response to the metabolic demands of lactation [5]. During periods of insufcient energy intake, oxidizable fuels in the diet are prioritized toward essential processes such as cell maintenance, circulation, and neural activity [6]. Homeorhetic controls in early lactation assure that body tissues, primarily adipose stores, are mobilized in support of milk production. Therefore, the dairy cow that is unable to consume enough energy-yielding nutrients to meet the needs of production and maintenance in early lactation will sustain yields of milk at the expense of body tissues. This poses a problem to reproduction, as delayed ovulation has been linked with energy status, e.g., energy deprivation reduced the frequency of LH pulses, impairing follicle maturation and ovulation [7]. This effect might be in part mediated by reduced concentrations of insulin and IGF-I; hepatic tissue of cows under negative energy balance is refractory to growth hormone (GH), and the uncoupling of the GH/ IGF system reduced concentrations of IGF-I [8]. Butler et al. [8] demonstrated that insulin re-established hepatic expression of GH receptor 1A, resulting in increased concentrations of IGF-I. These changes in hormone proles resulted in more estrogenic follicles

[9], which have been associated with resumption of ovulation [7] and improved oocyte quality [10]. Insulin appears to be a metabolic signal re-coupling the GH/ IGF system (improving energy intake), and increased concentrations of insulin might improve follicle competence and oocyte/embryo quality. The reduced embryo quality in nonsuperovulated lactating donor dairy cows [11] may be associated with changes in reproductive physiology, as a consequence of metabolism and nutrient demands by the mammary gland [5]. The negative effects of lactation and energy status on early embryo development are likely mediated by changes in oocyte quality, as both dairy and beef cows had reduced fertilization and embryo quality when lactating [3]. Increased concentrations of specic fatty acids (FA) in follicular uid during excessive body fat mobilization might impair fertilization and early embryonic development [12]; oocyte quality improved during the postpartum period, concomitant with improvements in energy balance and re-establishment of estradiol concentrations in follicular uid, particularly in lactating dairy cows receiving a diet with adequate energy concentration [10]. Therefore, endocrine changes associated with energy balance and lactation [5,7,12] and localized effects of metabolites in the follicular uid [12] may compromise oocyte and embryo competence in lactating donor cows. 3. Energy intake and superovulatory response Although there is controversy in the literature, follicle recruitment and superovulatory response can be altered by energy intake in cattle. Short-term changes in plane of nutrition increased recruitment of follicles in heifers with no effects on concentrations of FSH [13 16], which could increase ovulatory response after superstimulation [14]. Conversely, numerous other studies reported either no effect [17,18] or a decline in superovulatory response in nonlactating cows or heifers when energy intake increased above maintenance requirements [1922]. Increasing nutrient intake usually increased concentrations of insulin [1315,22,23] and IGF-I [23], attributed to increased production of rumen volatile FA, particularly propionate, and increased availability of glucose from hepatic gluconeogenesis [24]. These growth factors potentiate the effect of gonadotropins on follicular and luteal cells; differences in insulin and IGF-I concentrations may have accounted for improvements in follicle recruitment, despite similar concentrations of FSH [14]. Improvements in energy status in heifers have also been implicated in increased


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steroidogenesis of follicular cells [23]. It is unclear why nutrient intake above maintenance requirements might reduce superovulatory response in nonlactating donor cows. Increased feed intake enhanced catabolism of steroid hormones [5], expected to reduce inhibitory effects on gonadotropin secretion. Because FSH concentration usually did not change with altered energy intake [1315], an untested alternative explanation for improvements in superovulatory response with intake below maintenance requirements may be a change in sensitivity of tissues to gonadotropins, which in turn could enhance ovarian responses to FSH and LH. Nevertheless, the majority of studies indicated that short or long-term overfeeding of nonlactating cows or heifers reduced superovulatory responses [1922]. 4. Nutrient utilization by oocyte and embryos Prior to ovulation, the cumulusoocyte complex depends upon the surrounding uids for nutrition; therefore, alterations in composition of the follicular uid might affect oocyte competence and subsequent embryo development [1,12]. Furthermore, changes in uptake of nutrients by theca cells and transfer to granulosa cells might inuence nourishment of oocytes. For oocytes and embryos up to the morula stage, oxidative phosphorylation is the main pathway to generate energy-rich compounds for cellular metabolism. Presence of the glucose transporters GLUT-1 and GLUT-3 mediate the uptake of glucose by embryonic cells, which is then utilized to synthesize reducing equivalents such as NADPH during the pentose phosphate pathway. As embryos undergo a gradual shift from maternal mRNA to embryonic transcription, utilization of glucose and other carbon sources such as pyruvate and ATP synthesis increased [2], shifting energetic metabolism from oxidative phosphorylation to glycolysis. Nourishment of embryos before placentation depended on uptake from the histiotroph in the uterine lumen. In addition to glucose and carboxylic acids, embryos derive energy from stored glycogen and triglycerides; however, in mice, stored glycogen only provided energy for embryo metabolism for a few hours [25]. Triglycerides are the major lipid in bovine oocytes and lipolytic activity increased in bovine oocytes during maturation, suggesting that oocytes can utilize FA in stored triglycerides to generate ATP [26]. When FA oxidation was blocked with an inhibitor of mitochondrial transport, both oocytes and early embryos had impaired metabolism and reduced oxygen uptake [27]. In addition, amino acids such as glutamine and

threonine can be used to generate carboxylic acids succinate and a-ketoglutarate utilized for generation of ATP [25]. Although glucose and carboxylic acids are the major energy substrates for bovine oocytes and embryos, amino acids, FA stored as triglycerides and possibly FA present in the uterine lumen can be utilized to generate ATP for cell metabolism. Perhaps altering the availability of these substrates in the follicular uid, oviductal and uterine lumen by manipulating the diet of the donor cow might inuence oocyte and embryo development. 5. Inuence of energy intake on oocytes and embryos Insufcient energy intake in early lactation can delay cyclicity and compromise both follicle competence [7] and oocyte quality [10]. On the contrary, excessive energy intake and body weight gain in nonlactating donor cows or heifers compromised developmental competence of oocytes in vitro (Table 1) and embryo quality in vivo (Table 2). The impact of overfeeding energy on oocyte and embryo quality may be dependent upon the metabolic status of the donor cow, as the deleterious effects occured in overconditioned animals [28,29]. Interactions between supply of energy and degree of fatness may result in increased insulin responses; tissues of overconditioned cows are less responsive to insulin and develop insulin resistance, thereby reducing the uptake of glucose by cells. Despite dietary treatment and body condition, heifers experiencing hyperinsulinemia also produced oocytes and early embryos with reduced in vitro development [28]. Interestingly, in superovulated heifers gaining 0.95 kg/d and then subjected to either a more moderate weight gain (which reduced gain in body condition and plasma concentrations of insulin) or increased weight gain, the in vitro production and quality of blastocysts improved only with the moderate weight gain diet [30], suggesting that short-term restriction in nutrient intake might improve in vitro embryo production. Overfeeding usually increase glucose, insulin and IGF-I; pre-implantation exposure to high concentrations of IGF-I increased embryonic death in the mouse [31]. Although lower doses of exogenous GH usually increase conception rates in lactating dairy cows, a high dose of exogenous GH in nonlactating dairy cows increased plasma IGF-I and insulin, reducing pregnancy rates, and advancing development of embryos that survived [32]. Overfeeding might also expose oocytes and embryos to increased glucose concentrations; glucose was critical to metabolism of early embryos

J.E.P. Santos et al. / Theriogenology 69 (2008) 8897 Table 1 Effect of plane of nutrition in cattle on oocyte quality and developmental competence in vitro Plane of nutritiona Low Cows No. oocytes collected Viable oocytes (%) Cleavage (%) Blastocysts in in vitro culture (%) Heifers Oocytes fertilized (%) Cleavage (%) Blastocysts (% cleaved) Heifers Cumulus gradeb Cytoplasm gradeb Cleavage (%) Heifers Cumulus grade Cytoplasm grade Cleavage (%) Heifers Cleavage (%) No. excellent and good embryos No. blastocysts in in vitro culture Blastocyst cell number


P High 10 10.5 44.0 67.5 36.4 14 83 86 8 9 1.4 1.8 74.2 8 1.3 1.7 28.8 13 78.1 3.5 44 75.4


10 8.9 48.6 67.6 41.7 14 79 94 14 8 1.6 1.7 61.9 8 1.3 2.0 32.9 14 69.5 4.5 78 98.3

[16] NS c 0.10 NS NS [17] NS 0.01 NS [20] NS NS NS [20] NS NS NS [20] NS NS 0.01 0.001

Plane of nutrition varied according to study, but differed between low and high within experiment and/or study. Energy intakes per animal were: [16] low = 70% of energy requirement for maintenance, high = 170% of energy requirement for maintenance; [17] low = silage ad libitum, high = silage ad libitum +6 kg concentrate/d; [20] low = 9.6 Mcal of metabolizable energy/d, high = 28.6 Mcal of ME/d. b Grading scale of 15 scale (1 = excellent and 5 = degenerated). c P > 0.10.

[1,2], but chronic hyperglycemia reduced glucose uptake by embryonic cells and increased apoptosis [33]. Furthermore, glucose seemed to be more deleterious to female embryos, and a selective skew of gender was obtained when embryos were cultured in

high glucose concentrations [34]. Because overfeeding caused hyperinsulinemia [1315,22,23,2830] and might potentially increase exposure of embryos to glucose, which affected embryo survival [33], especially XX embryos [34], ET programs utilizing sexed

Table 2 Effect of plane of nutrition in cattle on embryo production and quality after superovulation Plane of nutritiona Low Cows No. total structures No. viable embryos Heifers No. excellent and good embryos No. transferable embryos Heifers No. total structures No. viable embryos 14 14.1 10.7 38 2.7 4.8 17 10.5 5.7 High 14 9.5 6.7 38 1.0 2.8 20 6.6 3.8 [18] 0.05 0.05 [21] 0.001 0.05 [22] 0.05 NS b P Reference

a Plane of nutrition varied according to study, but differed between low and high within experiment and/or study. Daily intakes per animal were: [18] low = 100% of energy requirement for maintenance, high = 180% of energy requirement for maintenance; [21] low = 3 kg of supplemental concentrate, high = ad libitum intake of concentrate; [22] low = 70% of energy requirement for maintenance, high = 170% of energy requirement for maintenance. b P > 0.10.


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spermatozoa to produce female calves may suffer to a greater extent than when unsexed or male-sorted spermatozoa are used. 6. Fatty acids and oocyte and embryo quality Lipids serve as source of energy and are critical components of the physical and functional structure of cells. Phospholipids play an important role in regulating the properties and activity of cell membranes. Changes in chain length, degree of unsaturation and position of the double bonds in the acyl chain of FA can have remarkable impacts on their function and may affect reproduction in cattle [35,36], although the potential implications on embryo quality and fertility are less clear [29,37]. A major impediment to the study of FA and reproduction in cattle is the inability to predict the availability of polyunsaturated FA for absorption in the small intestine. Because rumen microbial activity dramatically reduces the amount of polyunsaturated FA reaching the duodenum [38], it is difcult to determine the specic needs of reproductive tissues for specic FA. Nevertheless, fat supplementation improved fertility and effects differed with distinct FA sources [35,36]. The lipid content and composition of oocytes and embryos can inuence their competence, and the impact of lipids can be critical, particularly during cryopreservation. The FA composition of follicular uid, oocytes, and granulosa cells in dairy cows has been related to season of the year [39], which may explain differences in fertility of cows exposed to summer heat stress. In vitro development of oocytes collected in winter was greater than oocytes collected in summer; these changes were detected at different stages of development [39]. Moreover, phospholipids in follicular uid, granulosa cells and oocytes collected from cows in summer contained more saturated FA than in winter [39]. In summer, reduced feed intake and increased rumen retention time likely increase biohydrogenation of dietary unsaturated FA making less available for absorption. Reduced feed intake in summer may also lead to increased mobilization of stored lipids [24], 50% of which are saturated FA. As body temperature increases, cellular controls for incorporation of lipids into reproductive tissues may also alter selectivity for FA to avoid changes in membrane uidity, permeability, and function. When nonsuperovulated dairy cows were fed fat sources differing in FA prole from 25 d prepartum to 80 d postpartum, fertilization rate, number of accessory spermatozoa, and proportion of Grades 1 and 2 embryos

were all improved in cows receiving the more unsaturated FA [40]; however, supplementation of dairy cows with various sources of mono- and polyunsaturated FA from 3 wk prepartum to 100 d postpartum in summer did not inuence cleavage or in vitro embryo development [37]. These data illustrated the complexity of the effects of FA on embryo production, as in vitro results have not been corroborated by those in vivo [36]. Increasing fat supplementation with mostly saturated and monounsaturated FA improved in vitro development to blastocysts and increased the number of trophectoderm cells, despite no changes in oocyte quality and a reduced cleavage rate [41]. Embryos from gilts supplemented with a high-fat diet rich in linoleic acid (C18:2 n6) had an increased number of nuclei after cryopreservation compared to gilts fed a low-fat diet [42]. Similarly, ewes fed a diet supplemented with FA from sh oil for 13 wk had increased numbers of follicles and high quality oocytes, and improved integrity of oocyte membranes after cryopreservation compared with ewes not fed the diet [43]. It is unclear whether improved in vitro embryo production and viability after cryopreservation in some studies [4143] were caused by the increased fat intake or because of unsaturated FA; however, gilts fed diets with high linolenic acid (C18:3 n3) content had increased numbers of CL and oocytes and embryos with normal morphology [44]. Collectively, increasing the intake of unsaturated FA improves embryo quality and viability after cryopreservation, but results differed with the type of FA; inconsistency in response in cattle may be related to the supply of specic unsaturated FA for absorption in the small intestine. 7. Inuence of protein intake on oocytes and embryos The remarkable ability of ruminants to generate amino acids without dietary sources is related to rumen microbial digestion with production of protein from nonprotein nitrogen (N) sources [45]. Because of protein digestion by microbes, the rumen generates ammonia, which in turn is converted into urea after hepatic uptake [24]. Excessive concentrations of ammonia and urea in vitro have been associated with disruption of embryonic development [46,47]. Although it is unclear if disruptions observed in vitro reect oocyte and embryo responses in vivo, increasing circulating concentrations of urea N by manipulating the dietary energy and protein reduced conception rates of heifers [48], and embryos collected from lactating

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dairy cows fed a diet containing excess protein had reduced pregnancy rates in nonlactating recipients [49]. It has been suggested that increased concentration of ammonia and urea in the reproductive tract inuence embryo viability by altering follicular and oviductal environments [47,50]. Ewes fed a diet containing 3% urea produced Day 4 embryos that had reduced in vitro development to the blastocyst stage, but resulting blastocysts experienced accelerated development [50], which was also observed in vivo [51]. Conversely, Kenny et al. [52] observed that elevated concentrations of ammonia and urea did not inuence concentrations of glucose, lactate, K, Na, and Mg in the oviducts, but concentrations of Ca were reduced. One of the consistent effects of excessive dietary protein is a reduction in uterine lumen pH during the early luteal phase [48], which was associated with reduced conception rate. Similarly, infusion of urea increased plasma urea and reduced uterine luminal pH in dairy cows [53]. Altered secretion of endometrial cells exposed to high concentrations of urea and ammonia may reduce uterine pH, which affected embryo development [46] and fertility [48]. Maturation of oocytes in the presence of increasing concentrations of urea in vitro did not affect subsequent cleavage of zygotes, but seemed to have a negative effect on the proportion of blastocysts, whereas incubation of zygotes with the same concentrations of urea had no effect on embryo development [46]. However, reducing the pH of the culture medium to that observed in the uteri of cows fed excess protein abolished embryo development to the blastocyst stage. Results from in vivo embryo production studies in dairy cows were not consistent with the in vitro studies. When dry cows were superstimulated and received diets

with concentrations of protein and rumen degradable protein much higher than recommended for highproducing lactating dairy cows [24], no negative effects on embryo quality and viability were observed [54]. Similarly, excess protein (fed as urea) did not affect embryo quality in superovulated lactating cows [49]. When lactating diets differing in protein degradability were fed, the ration with higher rumen degradable protein tended to reduce the proportion of transferable embryos, even though the number of transferable embryos was not affected (Table 3) [55]. Although high urea and ammonia seemed to compromise in vitro embryo development [46,47] and, in some cases, inhibited growth and metabolism of oocyte-supporting granulosa cells [56], in vivo-produced embryos from dairy cattle fed excess protein often appear unaffected. Nevertheless, embryos of similar grade quality resulted in reduced pregnancy rate after transfer when they originated from cows consuming excess dietary protein [49]. As excessive dietary protein or non-protein N fed to donor cows might alter oviductal and uterine pH, it is recommended that donor cows should not be fed in excess of their needs for maintenance, growth and lactation. 8. Embryo toxicants Many other components of the diet of cattle have the potential to compromise fertilization and embryo development [50]. Gossypol, a toxic compound commonly present in rations containing cottonseed, adversely affected embryo development with as little as 5 mg/ mL in culture media [57], with the negative effects on gametes and embryos increasing in a dose-dependent manner [57,58]. Similarly, the negative effects of

Table 3 Effect of percentage crude protein (CP) and rumen degradable protein (RDP) on embryo quality in superovulated dairy cows CP (RDP) No. cows Embryos Transf* 15.7 21.9 12.3 27.4 16.0 16.1
a,b *

Oocytes Not transf 0.3 0.6 1.6 2.0 4.0 3.3 0.5 0.5 1.8 1.8 3.1 2.3

Transf (%)

DAPI** (%)


(ND) (ND) (60) (71) (73) (64)

12 11 22 22 19 19

3.4 5.0 4.0 4.9 4.5 5.5

82.0 83.0 49.7 54.0 44.2b 66.9a

ND*** ND 53.1b 66.7a ND ND

[49] [54] [55]

Within a column, means within an experiment without a common superscript differed (P < 0.10). Transferable embryos. ** Percentage of embryos negative for vital staining using 40 ,60 -diamidino-2-phebylindole, indicating intact cell membrane and lack of DNA staining. *** Not determined or specied in the study.


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gossypol on in vivo embryo quality and pregnancy in lactating dairy cows were dependent upon intake of free gossypol and its concentrations in plasma [5961]. In addition, embryos recovered on Day 4.5 from heifers fed 40 mg of gossypol/kg of body weight had retarded subsequent development in vitro [59]. When lactating dairy recipients received embryos from heifers fed either 0 or 12 g/d of free gossypol, embryos from heifers fed gossypol resulted in reduced pregnancy rates at 28 d and 42 d of gestation [62]. Similarly, lactating dairy cows fed diets high in free gossypol had marked increases in plasma gossypol, reduced conception rates, and increased pregnancy losses [61]. Therefore, it is prudent to limit and even eliminate the intake of gossypol in embryo donor cows. 9. General dietary guidelines for highproducing, lactating dairy donors As high-producing lactating dairy cows have the greatest nutrient needs for synthesis of milk and milk constituents, formulation of diets for oocyte or embryo production is a challenge; key components of the diet should be carefully evaluated. Diets of donor cows should contain adequate concentrations of ber carbohydrates to maintain rumen function in a manner that does not limit energy intake. Concentration of neutral detergent ber (NDF) in the diet as forage ber was more effective at maintaining rumen function than other nonforage ber sources [63]. Diets containing more than 35% NDF limit feed intake, thereby restricting energy and protein intake, exacerbating negative energy balance and body weight losses, all of which reduce fertility [7] and can lead to losses in production [24]. Nevertheless, total mixed rations with less than 28% NDF increase the risk for digestive disturbance and rumen acidosis [24]. To optimize feed and energy intake, and production of microbial protein, most rations should contain between 35 and 41% of the total dry matter as nonbrous carbohydrates [24,64]. Diets of high-producing dairy cows should contain a minimum of 2022% of the dry matter as physically effective NDF to maintain adequate rumen pH and milk fat content [65]. Furthermore, high-producing dairy cow diets should contain a minimum of 20% forage NDF [24], as the latter is the best predictor of rumen pH [63]. Therefore, diet formulation should combine the supply of rumen degradable carbohydrates and the concentrations of forage and physically effective NDF to maintain rumen health and optimize the supply of nutrients to the cow.

The cows tissues require an adequate supply of amino acids; however, the rumen microora has requirements for degradable protein to supply ammonia, amino acids, and peptides for microbial growth [24,64]. Until recently, crude protein was the main indicator of protein adequacy in rations for ruminants, in part because a large proportion of the amino acid needs are derived from microbial protein. Although protein of microbial origin can supply all amino acid needs of lactating cows producing up to 4500 kg of milk per lactation [45], increased output of milk in genetically superior cows has increased the requirements for amino acids; in cows producing >11,000 kg/year, only 5060% of the amino acid needs are from microbial protein in early- to midlactation [24,66]. In order to optimize production, but not increase the risk of N wastage and subsequent impairment of reproduction [48], diets for high-producing cows ought to contain between 16 and 18% crude protein [24], and when properly balanced, should not contain more than 17% crude protein after the rst 40 d of lactation [67]. Approximately 1011% of the total dry matter should be rumen degradable protein and, of that, 4050% rapidly degradable or soluble protein [64]. The undegradable protein fraction should have an amino acid prole that complements that of microbial protein [66,67]. Special attention should be paid to essential amino acids, particularly methionine and lysine in diets based on corn and alfalfa products, and histidine in diets based on grass silage and cereal grains. When diet formulation is based on meeting the needs of metabolizable protein and essential amino, dietary crude protein can be reduced [67], improving N use and minimizing the risk for increased urea and ammonia in reproductive tissues. Ruminants have evolved consuming diets marginal in FA content; however, fat supplementation in moderate amounts increased milk yields [35,68], and might inuence reproductive performance of dairy cows [35,36]. Increasing the energy density of the ration by feeding fat increased pregnancy rates of dairy cows [69]; however, FA supplementation has improved fertility despite energy intake, suggesting that supplementation of FA might be more appropriate than simply fat feeding [35,36]. Although FA can have positive effects on reproduction of cattle, fats can be detrimental to ruminal microbes and affect rumen digestion of ber and feed intake [68]. Furthermore, absorption of unsaturated FA can suppress appetite, which might reduce energy intake, particularly in early lactation. Typical diets contain <2% FA [68], and only moderate amounts of fat should be supplemented. When supplemental fat is based on sources rich in

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unsaturated FA, such as vegetable oils and oilseeds, or on other rumen-active fats (e.g. tallow and sh oil), total dietary fat should be limited to <5%, or FA to <4%. When fat sources inert to microbial function (e.g. saturated free-FA or calcium soaps) are fed, concentration of total fat in the ration may be increased up to 7%, or 56% FA. Increments above 7% of the dry matter as total fat do not pose any advantage to animal performance and should be avoided [24,68]. When selecting sources of FA, inert sources containing polyunsaturated FA such as linoleic and linolenic acids might benet oocyte and embryo quality, and resistance to cryopreservation [40,4244]. 10. Concluding remarks The exact mechanisms by which nutrition of the donor cow inuences oocyte and embryo quality and pregnancy after ET remain an enigma. Nutritional controls of reproduction are complex and mediated by a range of endocrine and metabolic signals that inuence follicle and oocyte competence and embryo quality. Despite the uncertainty of the mechanisms by which nutrients inuence oocyte and embryo production, diets should be formulated to optimize the supply of nutrients to meet the needs of different tissues according to the physiological status of the animal. Overfeeding energy and protein to cows is detrimental in ET and should be avoided. Concurrently, underfeeding postpartum cows can exacerbate the period of negative nutrient balance and compromise oocyte quality, fertilization and embryo development. Toxic compounds such as gossypol can hinder embryo development and reduce pregnancy rates following ET. Supplementation with lipids, particularly those rich in unsaturated FA have shown promise, but responses have not been consistent and in vitro results need to be validated with in vivo experiments evaluating pregnancy rate following ET. References
[1] Thompson J. The impact of nutrition on the cumulus oocytes complex and embryo on subsequent development in ruminants. J Reprod Dev 2006;52:16975. [2] Thompson JG. Dening the requirements for bovine embryo culture. Theriogenology 1996;45:2740. [3] Santos JEP, Thatcher WW, Chebel RC, Cerri RLA, Galvao KN. The effect of embryonic death rates in cattle on the efcacy of estrous synchronization programs. Anim Reprod Sci 2004;82 83:51335. [4] Breuel KF, Lewis PE, Schrick FN, Lishman AW, Inskeep EK, Butcher RL. Factors affecting fertility in the postpartum cow: role of the oocyte and follicle in conception rate. Biol Reprod 1993;48:65561.

[5] Wiltbank M, Lopez H, Sartori R, Sangsritavong S, Gumen A. Changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism. Theriogenology 2006;65:1729. [6] Wade GN, Jones JJ. Neuroendocrinology of nutritional infertility. Am J Regul Integr Comp Physiol 2004;287:127796. [7] Butler WR. Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. Livest Prod Sci 2003;83:2118. [8] Butler ST, Marr AL, Pelton SH, Radcliff RP, Lucy MC, Butler WR. Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A. J Endocrinol 2003;176:20517. [9] Butler ST, Pelton SH, Butler WR. Insulin increases 17 betaestradiol production by the dominant follicle of the rst postpartum follicle wave in dairy cows. Reproduction 2004;127:537 45. [10] Kendrick KW, Bailey TL, Garst AS, Pryor AW, Ahmadzdeh A, Akers RM, et al. Effects of energy balance on hormones, ovarian activity, and recovered oocytes in lactating Holstein cows using transvaginal follicular aspiration. J Dairy Sci 1999;82:173140. [11] Sartori R, Sartor-Bergfelt R, Mertens SA, Guenther JN, Parrish JJ, Wiltbank MC. Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. J Dairy Sci 2002;85:280312. [12] Leroy JLMR, Vanholder T, Mateusen B, Christophe A, Opsomer G, de Kruif A, et al. Non-esteried fatty acids in follicular uid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro. Reproduction 2005;130:48595. [13] Armstrong DG, McEvoy TG, Baxter G, Robinson JJ, Hogg CO, Woad KJ, et al. Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system. Biol Reprod 2001;64:162432. [14] Gong JG, Armstrong DG, Baxter G, Hogg CO, Garnsworthy PC, Webb R. The effect of increased dietary intake on superovulatory response to FSH in heifers. Theriogenology 2002;57:1591602. [15] Gutierrez CG, Oldham J, Bramley TA, Gong JG, Campbell BK, Webb R. The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers. J Anim Sci 1997;75:187684. [16] Martins AC, Ramos AF, Mollo MR, Pivato I, Camara JU, Carrijo LHD, et al. Inuence of high or low feed intake on in vitro embryo production in cattle (abstract). Acta Sci Vet 2006;34(Suppl 1):290. [17] Yaakub H, OCallaghan D, Boland MP. Effect of roughage type and concentrate supplementation on follicle numbers and in vitro fertilisation and development of oocytes recovered from beef heifers. Anim Reprod Sci 1999;55:112. [18] Bastos MR, Ramos AF, Driessen K, Martins AC, Rumpf R, Sartori R. Effect of nutritional ushing on the superovulatory response of crossbred cows (abstract). Acta Sci Vet 2007;35(Suppl 3):1242. [19] Staigmiller RB, Short RE, Bellows RA, Carr JB. Effect of nutrition on response to exogenous FSH in beef cattle. J Anim Sci 1979;48:118290. [20] Nolan R, OCallaghan D, Duby RT, Lonergan P, Boland MP. The inuence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers. Theriogenology 1998;50:126374. [21] Yaakub H, OCallaghan D, Boland MP. Effect of type and quantity of concentrates on superovulation and embryo yield in beef heifers. Theriogenology 1999;51:125966. [22] Mollo MR, Rumpf R, Martins AC, Carrijo LHD, Saueressig MG, Sartori R. Embryo production in superovulated Nelore heifers


J.E.P. Santos et al. / Theriogenology 69 (2008) 8897 under low or high feed intake (abstract). Acta Sci Vet 2007;35(Suppl 3):1241. Armstrong DG, Gong JG, Gardner JO, Baxter G, Hogg CO, Webb R. Steroidogenesis in bovine granulosa cells: the effect of shortterm changes in dietary intake. Reproduction 2002;123:3718. Nutrient requirements of dairy cattle. 7th ed. Washington, DC, USA: National Academic Press; 2001. Leese HJ. In: Milligan SR, editor. Metabolism of the preimplantation mammalian embryo, 13. Oxford Rev Reprod Biol; 1991. p. 3572. Cetica P, Pintos L, Dalvit G, Beconi M. Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro. Reproduction 2002;124:67581. Ferguson EM, Leese HJ. A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development. Mol Reprod Dev 2006;73:1195201. Adamiak SJ, Mackie K, Watt RG, Webb R, Sinclair KD. Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biol Reprod 2005;73:91826. Adamiak SJ, Powell K, Rooke JA, Webb R, Sinclair KD. Body composition, dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro. Reproduction 2006;131:24758. Freret S, Grimard B, Ponter AA, Joly C, Ponsart C, Humblot P. Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers. Reproduction 2006;131:78394. Pinto AB, Schlein AL, Moley KH. Preimplantation exposure to high insulin-like growth factor I concentrations results in increased resorption rates in vivo. Hum Reprod 2002;17:45762. Bilby TR, Guzeloglu A, Kamimura S, Pancarci SM, Michel F, Head HH, et al. Pregnancy and bovine somatotropin in nonlactating dairy cows. I. Ovarian, conceptus, and insulin-like growth factor system responses. J Dairy Sci 2004;87:325667. Moley KH. Hyperglycemia and apoptosis: mechanisms for congenital malformations and pregnancy loss in diabetic women. Trends End Met 2001;12:7882. Kimura K, Spate LD, Green MP, Roberts RM. Effects of Dglucose concentration, D-fructose, and inhibitors of enzymes of the pentose phosphate pathway on the development and sex ratio of bovine blastocysts. Mol Reprod Dev 2005;72:2017. Staples CR, Burke JM, Thatcher WW. Inuence of supplemental fats on reproductive tissues and performance of lactating cows. J Dairy Sci 1998;81:85671. Wathes DC, Abayasekara DRE, Aitken RJ. Polyunsaturated fatty acids in male and female reproduction. Biol Reprod 2007;77:190 201. Bilby TR, Block J, Amaral BC, Sa Filho O, Silvestre FT, Hansen PJ, et al. Effects of dietary unsaturated fatty acids on oocyte quality and follicular development in lactating dairy cows in summer. J Dairy Sci 2006;89:3891903. Doreau M, Ferlay A. Digestion and utilization of fatty-acids by ruminants. Anim Feed Sci Technol 1994;45:37996. Zeron Y, Ocheretny A, Kedar O, Borochov A, Sklan D, Arav A. Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles. Reproduction 2001;121:44754. Cerri RLA, Bruno RGS, Chebel RC, Galvao KN, Rutigliano H, Juchem SO, et al. Effect of fat sources differing in fatty acid prole on fertilization rate and embryo quality in lactating dairy cows (abstract). J Dairy Sci 2004;87(Suppl 1):297. [41] Fouladi-Nashta AA, Gutierrez CG, Gong JG, Garnsworthy PC, Webb R. Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows. Biol Reprod 2007;77:917. [42] Kojima T, Iwasaki T, Zeniya Y, Yoshino J, Totsukama K. Effect of dietary administration with linoleic acid and a-tocopherol on freezing tolerance in porcine embryos. J Reprod Dev 1996; 42:6772. [43] Zeron Y, Sklan D, Arav A. Effect of polyunsaturated fatty acid supplementation on biophysical parameters and chilling sensitivity of ewe oocytes. Mol Reprod Dev 2002;61:2718. [44] Kojima K, Zeniya Y, Aoyama T, Kondo A, Yoshino J. Dietary administration of fatty acids-enriched mold dried cell containing g-linoleic acid to female pigs improves ovulation rate and embryo quality in summer. J Reprod Dev 1997;43:1217. [45] Virtanen AI. Milk production of cows on protein-free feeds. Science 1966;153:16038. [46] Ocon OM, Hansen PJ. Disruption of bovine oocytes and preimplantation embryos by urea and acidic pH. J Dairy Sci 2003;86:1194200. [47] Sinclair KD, Kuran M, Gebbie FE, Webb R, McEvoy TG. Nitrogen metabolism and fertility in cattle. II. Development of oocytes recovered from heifers offered diets differing in their rate of nitrogen release in the rumen. J Anim Sci 2000;78:2670 80. [48] Butler WR. Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J Dairy Sci 1998;81:25339. [49] Rhoads ML, Rhoads RP, Gilbert RO, Toole R, Butler WR. Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows. Anim Reprod Sci 2006;91:110. [50] McEvoy TG, Robinson JJ, Ashworth CJ, Rooke JA, Sinclair KD. Feed and forage toxicants affecting embryo survival and fetal development. Theriogenology 2001;55:11329. [51] Berardinelli JG, Weng J, Burfening PJ, Adair R. Effect of excess degradable intake protein on early embryonic development, ovarian steroids, and blood urea nitrogen on days 2, 3, 4, and 5 of the estrous cycle in mature ewes. J Anim Sci 2001;79:1939. [52] Kenny DA, Humpherson PG, Leese HJ, Morris DG, Tomos AD, Diskin MG, et al. Effect of elevated systemic concentrations of ammonia and urea on the metabolite and ionic composition of oviductal uid in cattle. Biol Reprod 2002;66:1797 804. [53] Rhoads ML, Gilbert RO, Lucy MC, Butler WR. Effects of urea infusion on the uterine luminal environment of dairy cows. J Dairy Sci 2004;87:2896901. [54] Garcia-Bojalil CM, Staples CR, Thatcher WW, Drost M. Protein intake and development of ovarian follicles and embryos of superovulated nonlactating dairy cows. J Dairy Sci 1994; 77:253748. [55] Blanchard T, Ferguson J, Love L, Takeda T, Henderson B, Hasler J, et al. Effect of dietary crude protein type on fertilization and embryo quality in dairy cattle. Am J Vet Res 1990;51:9058. [56] Rooke JA, Ewen M, Mackie K, Staines ME, McEvoy TG, Sinclair KD. Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride. Anim Reprod Sci 2004;84:5371. [57] Brocas C, Rivera RM, Paula-Lopes FF, Mcdowell LR, Calhoun MC, Staples CR, et al. Deleterious actions of gossypol on bovine spermatozoa, oocytes, and embryos. Biol Reprod 1997;57: 9017.


[24] [25]













[38] [39]


J.E.P. Santos et al. / Theriogenology 69 (2008) 8897 [58] Zirkle SM, Lin YC, Gwazdauskas FC, Canseco RS. Effect of gossypol on bovine embryo development during the preimplantation period. Theriogenology 1988;30:57582. [59] Villasenor M, Coscioni AC, Galvao KN, Juchem SO, Santos JEP, Puschner B. Effect of gossypol intake on plasma and uterine gossypol concentrations and on embryo development and viability in vivo and in vitro (abstract). J Dairy Sci 2003;86(Suppl 1):240. [60] Coscioni AC, Villasenor M, Galvao KN, Chebel R, Santos JEP, Kirk JH, et al. Effect of gossypol intake on plasma and uterine gossypol concentrations and on embryo quality and development in superovulated Holstein dairy heifers (abstract). J Dairy Sci 2003;86(Suppl 1):240. [61] Santos JEP, Villasenor M, Robinson PH, DePeters EJ, Holmberg CA. Type of cottonseed and level of gossypol in diets of lactating dairy cows: plasma gossypol, health, and reproductive performance. J Dairy Sci 2003;86:892905. [62] Galvao KN, Santos JEP, Coscioni AC, Juchem SO, Chebel RC, Sischo WM, et al. Embryo survival from gossypol-fed heifers after transfer to lactating cows treated with human chorionic gonadotropin. J Dairy Sci 2006;89:205664.


[63] Allen MS. Relationship between fermentation acid production in the rumen and the requirement for physically effective ber. J Dairy Sci 1997;80:144762. [64] Hoover WH, Stokes SR. Balancing carbohydrates and protein for optimum rumen microbial yield. J Dairy Sci 1991;74: 363044. [65] Mertens DR. Creating a system for meeting the ber requirements of dairy cows. J Dairy Sci 1997;80:146381. [66] Santos FAP, Santos JEP, Theurer CB, Huber JT. Effects of rumen-undegradable protein on dairy cow performance: a 12year literature review. J Dairy Sci 1998;81:3182213. [67] Noftsger S, St-Pierre NR. Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efciency for milk production. J Dairy Sci 2003;86:95869. [68] Palmquist DL, Jenkins TC. Fats in lactating rations: review. J Dairy Sci 1990;63:114. [69] Ferguson JD, Sklan D, Chalupa WV, Kronfeld DS. Effects of hard fats on in vitro and in vivo rumen fermentation, milk production, and reproduction in dairy cows. J Dairy Sci 1990; 73:286479.