George E. Seidel

All life forms are under constant assault, resulting in an accumulation of damage within each individual, in both somatic and germline cells. The obvious causes are: (1) mutations from radiation, chemical reactions like peroxidation and errors in replicating genetic material; (2) injury due to environmental insults, such as chemical alteration of proteins by reactive oxygen species; (3) epigenetic errors, such as failure of appropriate maintenance methylation of cytosines of DNA; and (4) numerous other problems, including retroviral invasions, inflammation and unhealthy microbiomes. Collectively, these phenomena constitute aging and/or certain disease states. Nature has developed numerous mechanisms to counteract these problems, such as proofreading enzymes, ubiquitous antioxidants and apoptotic death of unfit cells. However, none of these is completely effective. Although individuals accumulate damage, species usually do not become increasingly damaged; however, this could be one of the mechanisms for eventual extinction or evolution to a different species, the apparent fate of essentially all species. Nevertheless, germline DNA appears to remain sufficiently pristine to maintain fairly stable phenotypes over many generations. How do species avoid accumulating damage when composed of individuals that do? One broad answer seems to be reproductive redundancy followed by elimination of defects through the death of gametes, embryos, fetuses, neonates and postpubertal individuals, with the culling pressure increasing as potential parents age. Another major force appears to be evolutionary pressure; individuals that best fit the environment out-reproduce those that fit less well. What is impressive is that older and older parents continue to have offspring that are nearly as pristine as those of younger parents, even though their germline cells have continued to age. Although the offspring of old parents are not as fit, on average, as those of young parents, differences are small and, in some species, compensated for by superior parenting with accumulated experience. To conclude, it appears that species do not age, even though they are composed of individuals whose somatic and germline cells have aged.

Contents: The objective of these experiments was to study bovine in vitro fertilization (IVF) conditions for blastocyst production using non‐sexed sperm (Experiment 1) and sexed sperm (Experiment 2). For Experiment 1, in vitro‐matured oocytes (N = 707) were allocated to a 2 × 3 × 4 factorial design: time of co‐incubation of gametes for fertilization (4 and 18 h), sperm dose (1, 0.33, and 0.11 × 10⁶ frozen–thawed sperm/ml, and sperm source (four bulls). Pronuclear status was evaluated for a subset. Experiment 2 (N = 2155 oocytes) was a 2 × 3 × 2 × 6 factorial design: sex of sperm (X and Y), sperm dose (1, 0.33, and 0.11 × 10⁶ frozen–thawed sperm/ml), and sperm‐sorting pressures (40 and 50 psi), replicated with sperm of six bulls. Presumptive zygotes were cultured 60 h in chemically defined medium‐1 (CDM‐1), and for 114 h in CDM‐2. For Experiment 1, pronuclear formation, cleavage and blastocysts rates were greater for 1, and 0.33 × 10⁶ than 0.11 × 10⁶ sperm/ml (72 and 62 vs 42%; 89 and 81 vs 58%; and 21 and 17 vs 9%, respectively; all p < 0.01); polyspermy was greater for 1, than 0.33 and 0.11 × 10⁶ sperm concentrations (24 vs 2 and 0%; p < 0.01). There were greater main effects (p < 0.01) of pronuclear formation (69 vs 48%), polyspermy (13 vs 4%), and cleavage (63 vs 54%), at 18 than at 4 h of co‐incubation of gametes (all p < 0.01). For Experiment 2, cleavage and blastocyst rates were greater for 1 × 10⁶ sperm/ml vs 0.33 and 0.11 (69%, 47%, and 30% cleavage and 30%, 14%, and 8% blastocysts) and 40 vs 50 psi (54% and 44% cleavage and 18% and 15% blastocysts) (p < 0.01). A marked bull by fertilization sperm dose interaction was found for cleavage (p < 0.05). The main conclusion was that the optimal sperm concentration for cleavage and producing blastocysts via IVF with sexed sperm was considerably higher and more variable among bulls than for unsexed sperm.

Genomic selection using single nucleotide polymorphisms (SNPs) is a powerful new tool for genetic selection. In cattle, SNP profiles for individual animals are generated using a small plastic chip that is diagnostic for up to 50 000 SNPs spaced throughout the genome. Phenotypes, usually averaged over offspring of bulls, are matched with SNP profiles of bulls mathematically so that animals can be ranked for siring desirable phenotypes via their SNP profiles. For many traits in dairy cattle, the rate of genetic improvement can be nearly doubled when SNP information is used in addition to current methods of genetic evaluation. Separate SNP analyses need to be developed for different populations (e.g. the system for Holsteins is not useful for Jerseys). In addition, the value of these systems is very dependent on the number of accurate phenotypes matched with SNP profiles; for example, increasing the number of North American Holstein bulls evaluated from 1151 to 3576 quadrupled the additional genetic gain in net merit from this approach. Thus, the available information will be insufficient to exploit this technology fully for most populations. However, once a valid SNP evaluation system is developed, any animal in that population, including embryos, can be evaluated with similar accuracy. Biopsying embryos and screening them via SNP analysis will greatly enhance the value of this technology by minimising generation intervals.

Over the next quarter century in North America, the following eventualities are likely for physiology and endocrinology research with agricultural animals. 1) Total funding adjusted for inflation will change little but will come less from public sources, and most of that will be in the context of human health. Much of the privately funded research will be herd specific and remain proprietary. 2) The numbers of MS, PhD, and postdoctoral students probably will decrease, but research in the context of credentialing will remain important. 3) Resources such as expanded databases in genomics and proteomics, and remarkable new tools such as small inhibitory RNA will continue to become available, likely at a faster rate than in the previous 25 yr. 4) The huge amounts of data from production agriculture will make agricultural animals ideal models for some kinds of basic research, such as studying fetal programming, resulting in synergy with more applied research. Most of these experimental animals will be in private production herds and flocks, even when work is publicly funded. 5) The trend toward more interdisciplinary research will continue, especially considering interactions among reproduction, health, nutrition, selective breeding, management factors, and societal concerns; reductionist research probing deeper into cellular and molecular mechanisms will remain important, as will whole-animal approaches. 6) Agricultural animals are a product of evolution plus selective breeding. Insights drawn from the former will aid progress in the latter. One focus of research in physiology and endocrinology will be understanding heterosis, inbreeding depression, and epigenetic effects as it becomes possible to manipulate and identify the allelic structure of individual animals. 7) Additional insightful concepts will evolve that will simplify thinking in some respects, such as the maternal to embryonic shift in transcribed RNA in early embryos; however, animal biology will turn out to be even more complex than most of us currently imagine. Copyright © 2009. . Copyright 2009 Journal of Animal Science

After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10-30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production.

Our objective was to determine if replacing glucose with fructose would decrease cytoplasmic lipid accumulation during culture of embryos with or without regulators of metabolism. In vitro‐produced bovine zygotes were cultured 60 hr in chemically defined medium‐1 (CDM‐1) plus 0.5% BSA and 0.5 mM fructose or glucose in Experiment 1, and glucose in Experiment 2. In both experiments, 8‐cell embryos were next cultured 135 hr in CDM‐2 plus 2 mM fructose or glucose in factorial combination with five treatments: (Experiment 1: control, 10% fetal calf serum (FCS), 0.3 µM phenazine ethosulfate (PES), 30 µM dinitrophenol (DNP), and PES + DNP), and (Experiment 2: control, PES, PES + DNP, and 1 and 3 µg/ml cerulenin (C1 and C3)). Day 7.5 blastocysts were stained with Sudan Black B to quantify cytoplasmic lipid droplets as small (SD, 6 µm). Blastocyst rates per oocyte were 22% (Experiment 1) and 15% (Experiment 2) higher (P < 0.05) for fructose than glucose. For Experiment 1, numbers of MD were lower for PES, DNP, and PES + DNP than control and FCS (P < 0.05). LD were lower for PES and DNP than control, and higher for FCS than all other treatments (P < 0.05). For Experiment 2, MD were lower (P < 0.05) for PES, and PES + DNP than C1, C3, and control. For LD, PES was lower (P < 0.05) than control, C1, and C3, but not different from PES + DNP. The only effect of hexose on lipids was that fructose resulted in fewer MD (P < 0.01) in Experiment 2. In conclusion, fructose produced more blastocysts than glucose, and PES reduced lipid accumulation. Mol. Reprod. Dev. 74: 1406–1418, 2007.