Reproduction in Domestic Animals

10 D Blache, GB Martin and SK Maloneyadaptability and redundancy of physiological systems,and because most systems are polygenic. In addition, inethical terms, the creation of knock-outs is costly for theanimals themselves because a proportion of geneticallymodified animals will not be viable and will have to bedestroyed before being part of an experiment (NuffieldCouncil on Bioethics 2005; NHMRC Animal WelfareCommittee 2007). Estimating the relative ratio betweencost and benefit for the use of genetically modifiedanimals is not an easy task and, to our knowledge, hasnot been addressed, as recognized in a recent reviewabout the use of transgenic farm animals to improveproduction (Bacci 2007).A different strategy for reducing inter-individualvariability, and thus addressing refinement and reduction,is the use of monozygotic twins (Biggers 1986).Twin studies have been used successfully in fields ofresearch such as health and immunology, but it does notseem to be as advantageous in reproductive biology. Insheep, for example, we have studied gonadotrophinresponses following an increase in nutrition, ovarianresponses to exogenous inhibin and the responses ofhypothalamo–pituitary axis to an opioid. For mostreproductive traits (ovulation rate, secretion of gonadotrophinsand sex steroids), randomly selected animalswere just as efficient for experimentation as geneticallyidentical twins (Celi et al. 2007). Monozygotic twinsshowed an advantage only for live weight and scrotalcircumference. In addition, the reproductive biotechnologiesthemselves need to address ethical questionsconcerning the overall imposition on animals neededto gain the result (Church 1988). Again, there is nodirect estimate of the reduction in the use of animalsfollowing cloning. On the contrary, the variancebetween clones in both behavioural and physiologicalparameters can be increased because of epigenetic andearly-life effects (Seidel 2001; Archer et al. 2003a,b).Nutritional input into reproduction and ethicalexperimentationThe relationship between the level of nutrition or, moregenerally, metabolic status and reproductive capacity isevident for all stages in the reproductive process:puberty, gamete production, conception and the survivalof embryos, fetuses and newborn (review: Robinsonet al. 2006). The responses can be rapid, with anacute increase in nutrient supply inducing multipleovulation in females (Vinoles et al. 2005) and anincrease in GnRH output in adult males (Zhang et al.2004). The responses can also be delayed in time, such asthe effect of peri-conception under-nutrition on thelength of gestation (Bloomfield et al. 2003). In addition,nutrition of the mother can affect reproductive processesin her offspring (‘fetal programming’), such as thenumber of Sertoli cells (Bielli et al. 2002) and length ofgestation and ovulation rate (Rae et al. 2001). Foetalprogramming is a relatively new field of investigationand many of the impacts of nutritional imbalance inutero on the reproductive physiology of the offspring arenot yet known (Rhind 2004). Thus, their potentialimpact on experimental design in, for example, a studydone with randomly selected animals, is currentlyimpossible to assess, although it is most likely that theyincrease between-animal variation.Therefore, if we are to ensure that the individuals usedin an experiment are homogenous, we need to considerthe metabolic status of not only the experimentalanimals but also their ancestors. Obviously, this phenomenonmight explain discrepancies among the outcomesof comparable experiments. We have beenconfronted with this problem in our own research intothe brain sites where steroids exert negative feedback onGnRH pulse frequency in mature Merino rams (Blacheet al. 1997). During a preliminary trial, done in WesternAustralia, implantation of oestradiol into the ventromedialnucleus of the hypothalamus decreased thefrequency of LH pulses. We then decided to repeat theexperiment with a larger number animals for statisticalvalidity, but in our collaborator’s laboratory in easternAustralia. The same treatment in the same brainlocation had no effect. A few months after this apparentfailure, we repeated the experiment a third time in thewest, and obtained the same results as in the first study.The contrary results could not be attributed to photoperiodor genotype. The answer came from anotherstudy, done at the same time, in which we demonstratedthat insulin was a very powerful stimulator of GnRHneuronal activity (Miller et al. 1995). When we measuredinsulin in samples from the steroid implantationstudies, we discovered that the concentration was aboutthree times higher in the eastern rams (31.4 ±1.4 ng ⁄ ml) than in the western rams (9.1 ± 0.6 ng ⁄ ml).This was not due to differences in their nutrition duringthe experiments, but because the eastern rams had abetter body condition (fatter) than the western ramsbefore the start of the experiment. We have thusconcluded, a posteriori, that the pre-experimental,nutritional history of the animals, and thus their tonicinsulin concentrations, prevented the oestradiol implantfrom inhibiting GnRH secretion in the eastern experiment.Thus, extensive knowledge of the history of theanimals can decrease the probability of conflictingexperimental outcomes. As we learn more about, forexample, the epigenetic effects of nutrition (Rhind 2004),this situation will become even more complex. Clearly,this is a particularly difficult issue for research done withfarm animals or wildlife.Animal emotion and ethical experimentationA major potential imposition on experimental animals is‘stress’ associated with the experimental protocol. Stressis an integral part of everyday life for all animals, withthe challenges of psychological or physical stressorsevoking the normal adaptive responses that maintainhomeostasis (Matteri et al. 1984). However, these sameresponses can not only interfere with an experimentaloutcome but also weigh heavily on the ‘cost’ side of thecost-benefit ratio. Thus, welfare can be improved byreducing any stress caused by experimental conditions,such as housing or the interactions between experimentalanimals and human experimenters. Our aim shouldbe to increase the freedom from fear and distress. This isvery relevant to studies in reproduction becausethe reproductive endocrine axis can be profoundlyÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag