Effects of Feeding OmniGen-AF ® during Superovulation on in Vitro Development of Embryos Recovered from Donor Beef Cows

Embryo quality is crucial when selecting embryos for transfer. Variation in quality may be attributed to poor oocytes, semen, stress, inflammation, and potential immune system dysregulation. OmniGen-AF ® (OG) feeding supports immune system function and animal health. Our laboratory recently reported lower percent degenerate embryos recovered and increased plasma progesterone in beef cattle donors fed OG during superovulation. In vitro development of embryos recovered from donor cows fed OG prior to collection is presented here. Embryos were recovered from 24 beef cows assigned to four treatment groups: 0 g OG/hd/d and 200 mg Folltropin ® -V (FSH) (0/200); 0 g OG/hd/d and 400 mg FSH (0/400), 56 g OG/hd/d, 200 mg FSH (56/200) and 56 g OG/hd/d and 400 mg FSH (56/400). Good to excellent quality early blastocysts were cultured for 8 d. and development through hatching, embryonic volume and plasminogen activator (PA) production were quantified. The complete protocol was repeated 90 - 120 d later as Replicate 2. Optimal development was observed by embryos recovered from 0/200 cows where percent blastocysts hatching was greater (P < 0.05) compared to 56/200 and 0/400 cows and embryonic volume was greatest (P < 0.05) in Replicate 1. However, percent blastocysts hatching from 0/200 cows was similar (P > 0.10) to 56/400 cows and embryos recovered from 56/400 cows in Replicate 1 produced more (P < 0.05) PA compared to all other groups. For cows superovulated with the standard 400-mg FSH dose, feeding OG supported in vitro embryo development similar to that observed for 0/200 cows.


Introduction
Assisted reproductive technologies are commonly used to improve overall herd genetics and female reproductive performance [1]. Embryo collection and transfer is a common assisted reproductive technology used in beef and dairy cattle. The key to the success of embryo transfer is recovering a high number of good to excellent quality embryos from donor cows for transferring to suitable recipients to achieve optimal pregnancy rates [2]. Numerous studies have been conducted employing nutritional strategies in feeding protocols during donor superovulation in attempts to increase both the number and quality of embryos recovered [3]. As an example, Yaakub et al. [4] fed dairy heifers increased concentrates during a superovulation protocol and recovered more embryos of poor quality compared to restricted-fed counterparts. Indeed, diet has been shown to impact gene expression in cattle embryos, specifically genes associated with blastocoel formation and oxidative stress [5] [6]. OmniGen-AF ® (OG; Phibro Animal Health Corporation, Teaneck, NJ) is a nutritional supplement shown to benefit overall animal health. OG is a specialty blend of ingredients that supports animal health during periods of stress, such as heat stress, by mitigating the negative physiologic effects associated with stress [7] [8] [9]. Feeding OG has also been shown to support immune system regulation during exposure to stressors [10] [11]. Dairy producers have reported a benefit in reproductive performance with the addition of OG into their herds [12] and incorporating OG into the ration of dairy cows has shown to reduce the number of days open [13]. Recently, our laboratory reported feeding OG to beef cow donors provided the beneficial effects of more transferrable embryos, fewer percent degenerate embryos and increased plasma progesterone on the day of embryo recovery [14]. The effects of feeding OG on donor embryo survivability have not been reported. Hormone dose is also an important factor to examine, as exogenous follicle stimulating hormone (FSH) can rescue follicles beginning to undergo atresia [15]. Reducing the FSH dose can lower the total number of embryos recovered but perhaps increase the number of transferrable embryos more likely to establish a pregnancy [16]. Good to excellent quality embryos may also produce more plasminogen activator (PA) during the hatching process [17]. PA is produced by blastocyst trophoblast and is proposed to participate in the breakdown of the zona pellucida via a zonalytic mechanism with uterine plasminogen [18]. Previous reports have demonstrated blastocysts completing the hatching process release significantly more PA than blastocysts failing to escape the zona pellucida [17]. Therefore, the objective of this research was to evaluate in vitro development and PA production as measures of viability in embryos recovered from donor beef cows fed OG and superovulated with 200 or 400 mg FSH.

Animal Housing and Feeding
Detailed descriptions of the animal housing and feeding and estrous synchronization, superovulation, artificial insemination and embryo collection protocols used in the study have been reported in Snider et al. [14] hence a brief description is presented here. Twenty-four cross-bred Angus cows were housed in a free stall barn at the Oregon State University Beef Center in Corvallis, OR with access to ad libitum grass hay and water. Cows were blocked for age, and randomly assorted into Control or OG-supplemented groups. All cows received a mixture of 0.2 kg ground corn and 6 oz molasses mix once a day. For cows fed OG, the supplement was hand-added to the corn/molasses mixture. All OG treatments were fed for 49 d with the superovulation protocol initiated on d 28 of the feeding period.

Estrous Synchronization, Superovulation and Artificial Insemination
Cows selected for Control or OG-feeding were randomly assigned to receive ei-

Embryo Culture and in Vitro Development
Early blastocysts of good to excellent quality were cultured singly in 15 hatched blastocyst (when the blastocyst is completely free of the zona pellucida).

Embryo Volume
After 192 h in culture, embryos were measured for volume using an ocular micrometer. Measurements were recorded either in diameter or length x width depending on the size and shape of the embryo and all measurements were recorded in micrometers. The volume of a prolate ellipsoid was used to determine embryo volume if the shape of the embryo deviated from that of a sphere ( Figure 1). The final value in the equation is given in cubic microns and was converted to nanoliters (1 nl = 1 × 10 6 cubic microns).

Plasminogen Activator Assay
PA concentrations in embryo-conditioned medium were determined using the caseinolytic agar gel assay described by Kaaekuahiwi and Menino [17]. To determine PA activity, 10 µl of conditioned medium or human urokinase standard

Statistical Analyses
Chi-square analysis was used to determine differences in percent embryos developing to the expanded, hatching and hatched blastocyst stages as a result of FSH dose, OG feeding and Replicate [20]. Analyses of variance (ANOVA) for 2 × 2 × 2 factorial designs were used to identify differences due to treatments in times required for embryos to develop to a specific cell stage and embryo volumes. Sources of variation in the ANOVA were FSH (200 or 400 mg), OG (0 or 56 g/hd/day), Replicate (1 or 2) and the interactions. Repeated measures ANOVA for a 2 × 2 × 2 factorial design were used to evaluate differences in PA concentrations during culture. Sources of variation in the ANOVA were FSH, OG, Replicate, time in culture (Time) and the interactions. In analyses where Replicate was not a significant effect data were pooled. If significant effects were observed in the ANOVA differences between means were evaluated using the Fisher's least significant differences procedures. All analyses were performed using the NCSS statistical software program (Number Cruncher Statistical System; 2007, Jerry Hintze, Kaysville, UT).

Results
Fifty-three and 44 good to excellent quality early blastocysts recovered from cows in Replicates 1 and 2, respectively, were selected for culture (Table 1). Three to five cows within each treatment group and replicate contributed to the pool of embryos. Replicate did not affect (P > 0.10) percent embryos developing to the expanded (Replicate 1, 98% compared with Replicate 2, 96%), hatching (Replicate 1, 92% compared with Replicate 2, 93%) and hatched blastocyst (Replicate 1, 87% compared with Replicate 2, 86%) stages. Development to the expanded blastocyst stage was similar (P > 0.10) for embryos recovered from cows superovulated with either dose of FSH (200 mg, 97% compared with 400 mg, 97%) and fed either 0 or 56 g OG (0 g, 98% compared with 56 g, 98%) ( Figure 2). Percent embryos initiating hatching was similar (P > 0.10) for embryos recovered from cows superovulated with either dose of FSH (200 mg, 95% compared with 400 mg, 92%) and fed either 0 or 56 g OG (0 g, 91% compared with 56 g,      (Figure 4). The FSH X OG X Replicate interaction was also found to be significant where PA production was greatest (P < 0.05) from embryos recovered from 56/400 cows in Replicate 1 compared to all other treatments in Replicates 1 and 2 ( Figure 5).
Plasminogen activator production differed (P < 0.05) by Time in culture and a significant Replicate X Time interaction was observed ( Figure 6). Peak PA production a,b Means without similar superscripts differ (P < 0.05).   (P < 0.05) was measured during 72 -120 h of culture and production was greater (P < 0.05) by embryos recovered in Replicate 1 at 48 -192 h compared to Replicate 2 ( Figure 6).

Discussion
Embryo quality can be affected by many variables during the superovulation process [1]. Transferring an embryo of optimal quality is critical to ensure successful hatching, attachment and pregnancy establishment in recipient cows [2] Certain factors involved with superovulation, such as FSH dose, handling of donor cows and overall animal health can contribute to a stress response affecting embryo quality [21] [22]. In a recent report from our laboratory, feeding donor beef cows OG during superovulation provided more transferrable embryos and  decreased percent degenerate embryos recovered [14]. Results presented in this report demonstrate how feeding OG, a health-supporting supplement, in con- missing the critical window for signaling pregnancy [24]. From these results there appears to be a benefit for embryos recovered from donor cows superovulated with 400 mg FSH and receiving OG feeding. Perhaps modulation of the immune system or inflammation by feeding OG is indirectly affecting follicular dynamics or uterine physiology [9] [25].  [17]. It is also possible the pool of embryos generated in the first cycle of superovulation were more robust than the second. This is somewhat evidenced by the greater mean volume observed in Replicate 1 compared to 2 and may be due to the quality of the selected oocyte pool. There is also an apparent incongruity with embryonic volume and PA production as embryos with the greatest volume did not have the greatest PA production. Kaaekuahiwi and Menino [17] reported significant correlation coefficients for bovine embryo diameter and cell number with PA production of 0.40 and 0.35, respectively, hence some deviation from this relationship would not be unexpected. In this same study, the correlation coefficient for bovine embryo diameter with cell number was 0.79 [17]. Clearly, feeding OG to cows superovulated with 400 mg had an impact on overall development, including PA production.
Overall, the results suggest a positive effect of feeding OG to cows superovulated with 400 mg FSH on early embryonic development when evaluated in vitro. Whether this benefit extends to a greater likelihood of pregnancy establishment in recipients requires additional research as well as determining how OG induces its biological effect. Feeding cows OG has been linked to lowering the stress response and supporting immune function [8] [11] [26]. Perhaps through these mechanisms, OG is exerting positive effects on follicular maturation and embryo development during superovulation protocols in donor cows.

Conclusion
Development in vitro was greatest by embryos recovered from cows fed 0 g OG and treated with 200 mg FSH suggesting the most favorable treatment regimen. and ovulation may include ovarian damage due to inflammation and tissue scarring, down-regulation of hormone feedback due to high hormone concentrations and shifts in uterine secretions resulting in an intrauterine environment that is unsupportive for embryo survival or growth. Feeding OG may provide immune and physiological support during this process for ovarian repair and uterine environment modulation to support embryo survival and growth. Further research is needed to elucidate the physiologic mechanisms and target organs defining how feeding OG provides a beneficial effect on follicular maturation and embryo development. Overall, the results support a potential benefit for embryo viability when donor cows are fed OG during a superovulation protocol using the standard dose of 400 mg FSH.