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ANIMAL PRODUCTION |
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* Department of Animal and Range Sciences,
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Cell Biology Center, and
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Center for Nutrition and Pregnancy, North Dakota State University, Fargo 58105
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: assisted reproduction • embryo • in vitro fertilization • nutrition • sheep
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Guler et al., 2000; Rizos et al., 2002) and diet manipulation of donor ewes (O’Callaghan et al., 2000; Lozano et al., 2003; Peura et al., 2003) and cows (Yaakub et al., 1999; Sinclair et al., 2000; Armstrong et al., 2001).
Nutritional status is a major factor influencing an animal’s ability to reproduce (Robinson, 1990; Webb et al., 1999; O’Callaghan et al., 2000). Nutrition has a significant impact on numerous reproductive functions including hormone production, fertilization, and early embryonic development (Boland et al., 2001; Armstrong et al., 2003; Boland and Lonergan, 2005). Nutritional status has been correlated with embryo survival and is a key factor influencing efficiency in assisted reproductive technologies (Armstrong et al., 2003; Webb et al., 2004). Conflicting results have been reported for the effects of low or high energy diets on oocyte quality and early embryonic development in ruminants including sheep (Boland et al., 2001; Papadopoulos et al., 2001) and cows (Nolan et al., 1998; Yaakub et al., 1999; Tripp et al., 2000). Therefore, additional study should clarify the effects of nutritional plane on oocyte quality and early embryonic development.
We hypothesized that plane of nutrition would alter oocyte quality as measured by rates of fertilization and early embryonic development in vitro. Therefore, the aim of the current study was to evaluate effects of nutritional plane (control vs. underfed) on follicular development, in vitro fertilization (IVF), and early embryonic development in FSH-treated ewes.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Eighteen (2- to 3-yr-old), western range (predominantly Targhee and Rambouillet) ewes of similar BW (47.0 ± 1.5 kg; mean ± SEM) and BCS (3.36 ± 0.04) were used in the study. Body condition score was determined using a 5-point scale (1 = extremely thin to 5 = extremely fat). Ewes were housed and fed in individual pens (0.86 x 1.47 m) at the ANPC under 14 h of darkness and 10 h of light at 12° C with free access to water and mineral supplements. Ewes were randomly divided into 2 groups (n = 9/each). Both groups were adapted during the first 4-wk (acclimation) period to 2 planes of nutrition (control and underfed). During the next 4 wk (second period), control ewes (adequate) were fed to maintain BW, whereas underfed ewes (restricted) received 60% of the daily dietary allocation for control ewes (see nutritional management section). Once each week, throughout the duration of the experiment, ewes were weighed and BCS was determined.
To synchronize estrus, 30 d after initiation of the nutritional treatment, chronogest (progestagen) sponges (30 mg of flugestone acetate/sponge; Intervet, UK) were inserted into the vagina for 14 d. Through the use of vasectomized rams, estrus (d 0) was detected 40 to 48 h after sponge withdrawal. Ewes received twice daily (morning and evening) injections with FSH-P (Sioux Biochemical, Sioux Center, IA) on d 13 (5 units/ injection) and 14 (4 units/injection) after estrus, as described previously (Stenbak et al., 2001). On d 15 of the subsequent estrous cycle, ewes were ovariectomized (Luther et al., 2005). The study was initiated during the normal breeding season in September and finished in December.
Nutritional Management
After arrival and a 3-d adaptation to individual pens and pelleted diets, ewes were allocated randomly to 2 nutritional groups, as just described. The diet contained (% of dietary DM): dehydrated beet pulp, 36.5%; dehydrated alfalfa, 20.3%; corn, 24.2%; soy hulls, 16%; and soybean meal, 3.0%. The pelleted (0.48-cm diameter) diet, which was prepared and analyzed on site, supplied 2.4 Mcal of ME and 130 g (13%) of CP per kg of diet (DM basis) and was offered in 1 equal portion daily. During the first 4-wk (acclimation) period, control ewes received 1,000 g/d and underfed ewes received 600 g/d (DM basis). The acclimation period transitioned ewes into the second 4-wk period, which again contained 2 different planes of nutrition. After the first 4-wk acclimation period, dietary intake was reduced in control ewes so that BW and condition were maintained (adequate; fed 720 g/d of diet DM) and underfed ewes were restricted to 60% of controls (restricted; fed 432 g/d of diet DM). Ewes were then maintained on their respective planes of nutrition for 4 wk, until oocyte collection.
Oocyte Collection
After ovariectomy, the ovaries were immersed in PBS and transported to the laboratory in an incubator at 39° C. The number of visible small (
3mm) and large (>3mm) follicles on each ovary was determined, and cumulus oocyte complexes were isolated by opening each visible follicle with a scalpel blade and flushing it 2 to 3 times with oocyte collection medium (Grazul-Bilska et al., 2003, 2005; Luther et al., 2005). Under a stereomicroscope, cumulus oocyte complexes were recovered from each dish and transferred to a petri dish containing fresh collection medium without heparin. Cumulus oocyte complexes were then evaluated and categorized as healthy or atretic based on their morphology (Thompson et al., 1995). All cumulus oocyte complexes were then washed 3 times in maturation medium [TCM-199 containing 10% fetal bovine serum, ovine FSH (5 µg/mL; oFSH-RP-1; NIAMDD-NIH, Bethesda, MD), ovine LH (5 µg/mL; oLH-26; NIADDK-NIH), estradiol-17ß (1 µg/mL; Sigma, St. Louis, MO), glutamine (2 mM; Sigma), sodium pyruvate (0.25 mM; Sigma), epidermal growth factor (10 ng/mL; Sigma), and penicillin/streptomycin (100 units of penicillin/mL and 100 µg of streptomycin/mL; Gibco, Grand Island, NY)].
In Vitro Maturation
Oocytes were matured in vitro in maturation medium for 24 h at 39° C in 5% CO2 and 95% air, followed by cumulus cell removal using 1% (wt/vol) hyaluronidase (Type I, Sigma) in PBS. Oocytes were again evaluated for health based on morphology. Oocytes classified as healthy were used for IVF and were transferred to equilibrated fertilization medium consisting of synthetic oviductal fluid prepared in our laboratory (Stenbak et al., 2001) and containing 2% heat-inactivated serum collected from sheep on d 0 to 1 of the estrous cycle (Grazul-Bilska et al., 2003, 2005; Luther et al., 2005).
In Vitro Fertilization and Embryo Culture
Frozen capacitated semen pooled from 4 Hampshire rams was thawed, and viable sperm were separated using the swim-up technique (Grazul-Bilska et al., 2005). The sperm (0.5 to 1.0 x 106 sperm/mL) were added to the IVF medium containing the oocytes and incubated for 18 h at 39° C, 5% O2, 5% CO2, and 90% N2. The presumptive zygotes were then washed 3 times with culture medium without glucose [synthetic oviductal fluid supplemented with BSA, glutamine, MEM nonessential amino acids, BME amino acids (Sigma), and penicillin/streptomycin] and cultured in the same medium for 24 h at 39° C, 5% O2, 5% CO2, and 90% N2 (Grazul-Bilska et al., 2003, 2005). After the 24-h incubation, dishes were evaluated to determine the number of cleaved oocytes. Embryos were then transferred to culture medium containing glucose (1.5 mM). After an additional 48-h incubation, the developmental stage was evaluated and embryos were transferred to fresh culture medium with glucose. Rate of cleavage (number of cleaved vs. noncleaved oocytes), and rate of early embryonic development (time and percentage reaching morula or blastocyst stages) were evaluated every second day during 8-d culture.
Evaluation of Maturation Status of Oocytes
Three days after IVF, all noncleaved oocytes were separated from embryos, and sperm was removed by repeated pipetting with a micropipette of 150-µm diameter. Naked oocytes were then fixed in methanol, followed by staining with 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Eugene, OR) and evaluation of the maturation status under epifluorescent microscopy. Oocytes in the germinal vesicle stage, containing diplotene chromatin, were considered to be immature. Oocytes in metaphase II demonstrated extrusion of the first polar body and were considered to be mature (Luther et al., 2005; Pant et al., 2005).
Statistical Analysis
To compare changes in BW, ADG, and BCS during the experiment, the number of follicles and oocytes, and oocyte quality variables (e.g., the rates of cleavage, and morula and blastocyst formation) for control and underfed ewes, data were analyzed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). For all variables, the model included only nutritional treatment. Absolute values for BW and BCS were analyzed using a repeated measures design (Proc Mixed of SAS). The model contained treatment, week of measurement, and treatment x week interaction. Ewe within treatment served as the error term to test for treatment responses. Compound symmetry was used as the covariate structure. Means were separated using the method of least significant difference. When treatment x week interactions were present (P < 0.05), the interaction means were tested. In addition, data for the percentage of oocytes cleaved and the rate of morula and blastocyst formation (%) were analyzed by 
2. Simple correlations between BW or BCS and blastocyst formation variables were determined using Proc Corr of SAS.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). A treatment x time interaction was detected (P < 0.05) for both BW and BCS; therefore, effects of treatment were examined within week, and differences among weeks were evaluated within treatments. By the end of the first 4-wk acclimation period, BW of control ewes increased (P < 0.001) compared with initial BW and then remained unchanged at wk 4 and 8 of the experiment (Figure 1A). By the end of the first 4-wk acclimation period, BW of underfed ewes was similar to initial BW and then was decreased (P < 0.01) at wk 8 of the experiment (Figure 1A). Body weight of underfed ewes was lower (P < 0.05) than control ewes at wk 4 and 8 of the experiment (Figure 1A). Magnitude of change in BW was less (P < 0.001) from wk 0 to 4 and greater (P < 0.001) from wk 4 to 8 for underfed compared with control ewes (Table 1). Average daily gains were lower (P < 0.001) for underfed compared with control ewes during the first 4 wk but not during the last 4 wk of experiment (Table 1). However, ADG for the entire 8-wk experiment was greater (P < 0.001) for control compared with underfed ewes (0.19 vs. –0.05 ± 0.03 kg). When compared with initial BW, control ewes gained 10.6 ± 2.1 kg, but underfed ewes lost 2.6 ± 1.2 kg over the 8-wk experiment.
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). By the end of the first 4-wk acclimation period, BCS of control ewes increased (P < 0.01) compared with initial BCS and was unchanged until the end of the experiment (Figure 1B). At wk 4, BCS of underfed ewes was similar to initial BCS and then decreased (P < 0.01) at wk 8 of the experiment (Figure 1B). Body condition score of control ewes was greater (P < 0.001) than underfed ewes on wk 4 and 8 of the experiment (Figure 1B). Change in BCS was lower (P < 0.003) on wk 4 but greater (P < 0.03) on wk 8 for underfed compared with control ewes (Table 1). During 8-wk experiment, BCS increased by 0.42 ± 0.1 for control ewes but decreased by 0.22 ± 0.1 for underfed ewes.
Mean number of visible follicles, number of large and small follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, and the number of cleaved oocytes per ewe were similar for control and underfed groups (Table 2). The cleavage rates, number of blastocysts, and the rates of blastocyst formation were greater (P < 0.001 to 0.006) for control compared with underfed ewes (Table 2). The total number of morula and the rate of morula formation tended (P < 0.093) to be greater for control compared with underfed ewes (Table 2). Total number of oocytes used for IVF was 123 for control and 124 for underfed ewes. Maturation rate evaluated for noncleaved oocytes was similar for control and underfed ewes (Table 2). Overall, of the total number of oocytes used for evaluation of maturation status (6.4 ± 1.1/ewe), the mean number and the percentage of matured oocytes were 4.7 ± 0.6 and 75.1 ± 7.1%, respectively. Number and percentage of blastocysts were positively correlated with the final BW (0.524, P < 0.02 and 0.650, P < 0.003, respectively) and BCS (0.592, P < 0.01 and 0.511, P < 0.03, respectively).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In previous studies, for mature ewes fed a low energy diet (approximately 0.5 to 0.6 times maintenance energy requirement) for 3 to 4 wk, decreased BCS (from 2.61 to 2.1 and from 2.5 to 2.33; Abecia et al., 1999 and Lozano et al., 2003, respectively) was observed along with decreased cleavage rates, number of good quality embryos, or the rates of pregnancy. On the other hand, ad libitum feeding of ewes for approximately 3 wk resulted in enhanced BCS (from 2.58 to 2.7) but lower superovulation responses, lower number of good quality oocytes and embryos, and a greater percentage of poorly developed embryos in mature ewes (Lozano et al., 2003). In addition, Snijders et al. (2000) demonstrated that rates of cleavage and blastocyst formation from oocytes derived from cows with BCS 1.5 to 2.5 were less (70.4 vs. 77.4% and 6.8 vs. 11.4%, respectively) than those from cows with BCS 3.3 to 4.0 during the first or third lactation. These data indicate that decreased BCS may be associated with decreased oocyte quality measured by rates of in vitro fertilization and early embryonic development in sheep and cows. This suggests that BCS can be used to predict successful embryonic development. However, determination of BCS is relatively subjective and may vary from study to study.
In the present experiment, nutritional plane had no effect on the number of ovarian follicles. Peura et al. (2003) reported for adult ewes that low (0.7x ) or high (1.3x ) maintenance diets for 3 to 5 mo before FSH-induced superovulation did not affect ovulation rates. Moreover, superovulatory responses after FSH treatment were not affected by 0.5x, 1.0x, or 1.5x maintenance diet fed during the periconception period in adult ewes (Kakar et al., 2005). These data indicate that these specific nutritional treatments did not affect follicular development measured by the number of follicles or ovulations in sheep. The number of follicles was similar in lactating dairy cows (not treated with FSH) receiving low (1.52 Mcal of NEl/kg of DM) or high (1.78 Mcal of NEl/kg of DM) energy diet for approximately 25 wk postpartum (Kendrick et al., 1999) and in yearling beef heifers (not treated with FSH) fed ad libitum or 0.75 x ad libitum for 100 d (Tripp et al., 2000). On the other hand, FSH-treated beef heifers fed a low-energy (9.6 Mcal/kg of ME/d) diet for 17 to 19 d had more follicles than cows fed a high-energy (28.6 Mcal/kg of ME/d) diet (Nolan et al., 1998). In addition, in the current study we observed similar maturation rates for nonfertilized oocytes in both groups. Thus, these results show that nutritional treatments had no effect on the number of visible follicles and the rate of oocyte maturation in sheep, but this was not true in cows. Moreover, the number of follicles in the current study was similar to that previously reported for FSH-treated mature ewes fed a maintenance diet during the normal breeding season and seasonal anestrus (Stenbak et al., 2001; Grazul-Bilska et al., 2003; Luther et al., 2005). However, the rate of maturation for noncleaved oocytes in the current study was 10 to 20% lower than reported for ewes fed a maintenance diet during seasonal anestrus (Luther et al., 2005).
Cleavage rates were lower for underfed (63%) compared with control (79%) ewes in our study. Papadopoulos et al. (2001) showed that cleavage rates were decreased (from 88 to 66%) in ewes fed a low energy (0.5 x maintenance energy requirements) diet in comparison with a high energy (2 x maintenance energy requirements) diet for 28 d. Similar to our results for underfed ewes, low cleavage rates, 51 and 35%, were observed for ewes underfed (0.5 x maintenance energy requirements) and overfed (ad libitum intake) for approximately 24 d, respectively (Lozano et al., 2003). This indicates that inadequate diet (e.g., underfeeding or feeding ad libitum) affects oocyte quality measured by IVF rates in sheep. In addition, rates of cleavage similar to cleavage rates in our control group were reported for mature sheep fed a maintenance diet (Watson et al., 1994; Ledda et al., 1997; O’Brien et al., 1997). Thus, the rates of fertilization may be influenced by different nutritional regimens under which oocytes were developed in the maternal environment.
In the current study, number of blastocysts and rate of blastocyst formation were lower for underfed ewes compared with control ewes. In contrast, Lozano et al. (2003) demonstrated that restricted diets (0.5 x maintenance diet for about 24 d) did not affect the rates of blastocyst formation in mature sheep. In addition, a low-calorie (0.6 x maintenance) diet fed for about 2 wk increased viability, protein synthesis index, and number of nuclei in the blastocyst developed from embryos produced in vivo and then cultured in vitro compared with those produced in mature ewes fed maintenance or a high calorie diet (McEvoy et al., 1995). Similarly, a greater number of cells in blastocysts produced in vivo was observed for ewes fed low-calorie diets (0.5 x maintenance diet) compared with ewes fed 1 x or 1.5 x maintenance diets during the periconception period (Kakar et al., 2005). Data from these studies indicate that ewes can respond to acute changes in nutrition during the periconception period, resulting in changes of embryonic development. Moreover, supplementation with urea to the diet with low energy (0.5 x maintenance) did not affect blastocyst cell number and blasto-cyst hatching rate in sheep (Papadopoulos et al., 2001). For yearling beef heifers, restriction of dietary energy (75% of ad libitum fed) did not affect the rate of blasto-cyst formation (Tripp et al., 2000). Therefore, it seems that the level of feed restriction, length of restricted feeding, or both reported in some studies was not severe enough to induce a negative impact on the rate of blasto-cyst formation, which was observed in the current study. Moreover, effects of nutritional treatment on blastocyst formation may also depend on specific diet composition and breed.
Numerous experiments indicate that nutrition has direct effects on some reproductive function by affecting hormone production (O’Callaghan and Boland, 1999; Lucy, 2003; Hunter et al., 2004). For example, for underfed or overfed mature ewes with enhanced or decreased blood progesterone concentrations, respectively, altered oocyte and embryo quality were observed (Lozano et al., 2003). This indicates that effects of nutrition on oocyte and embryonic development may be indirectly linked through regulation of hormone secretion. In the current study, peripheral blood hormone concentrations were not evaluated. Therefore, future studies should further define association between hormone concentrations and oocyte quality.
Effects of nutrition on oocyte and embryonic development may reflect the general energy balance (e.g., maintenance diet vs. low or high energy diets) but also can be attributed to the specific nutrients in diets, such as vitamins, minerals, and other supplements (Wrenzycki et al., 2000). For example, Tarin et al. (1998) observed that supplementation with a mixture of vitamins C and E to the maternal diet enhanced the number of ovulations but did not affect rates of cleavage or blasto-cyst formation in mice. Moreover, McEvoy et al. (1997) demonstrated that a high concentration of urea in diet fed for 12 wk increased embryo mortality and decreased pregnancy rates after embryo transfer in mature sheep. Additional studies should determine which nutritional factors affect oocyte quality.
An alternative interpretation of the present data set could be that increased BW and BCS in the control group due to plane of nutrition before IVF resulted in elevated cleavage rate, blastocyst, and rate of blastocyst formation. However, based on previous data from our laboratory and others (discussed earlier), we think that our primary interpretation of the data is most likely correct. In either case, current data clearly point to the need for additional studies evaluating effects of nutritional management on IVF and early embryonic development.
In conclusion, number of developing follicles, number of recovered oocytes, number and percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar in our 2 treatment groups. However, ewes restricted to 60% of the control diet had decreased rates of fertilization and blastocyst formation. These data indicate that donor animals likely require a specific nutritional management to provide good quality oocytes for assisted reproductive technology. Nutrition of donor animals seems to be a key component affecting development of oocytes and the preimplantation embryo. We restricted total dietary intake in the current study, but future investigations that address specific dietary nutrient composition should provide insight into the underlying mechanisms associated with changes in efficiency of in vitro embryo production.
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Footnotes |
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2 Corresponding author: Anna.Grazul-Bilska{at}ndsu.edu
Received for publication July 15, 2005. Accepted for publication January 22, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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production in vitro, interferontau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes. Theriogenology 52:1203–1213.[Medline]
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