• MammalianReproduction

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Different endocrinological aspects of mammalian reproduction

Introduction: Presentation of Endocrinological reproduction

Reproduction, by definition, is the biological process by which new individual organisms (« offspring ») are produced from their parents by combining the genetic material of 2 organisms (Wikipedia). Reproduction is one of the three primary needs of mammalian survival and has a role in supporting evolution. The neuroendocrine center, located in the Hypothalamus, acts as the major regulatory center of reproductive processes by mean of the action of hypothalamic GnRH cells that are going to stimulate all the hormones necessary to the control of reproduction. In the mammalian female, those hormones function by mean of a cycle which can be very different depending of species. Endocrinological regulation is a need in reproduction as it controls sexual maturity, normal sexual cycles, pregnancy, parturition and stability of the whole sexual mechanism.

How can Endocrinology affect or improve Reproduction in some mammalian species ?

Part III – The role of endocrinology in animal production : the dairy cow

Nowadays, the actual goals and roles of dairy farming is to produce more and more due to oppressing increase in population and demand. Heifers need to conceive earlier and get better production performances in average. To answer those actual questions, a perfect and well-managed knowledge of sexual endocrinological hormones is essential. This knowledge can be reinforced by the development of technologies such as Ultrasonography (monitoring follicular growth) and genomic technologies.

1) Effects of artificial endocrinology on early puberty and pregnancy in dairy heifers

Puberty is defined as « when ovulation is accompanied by visual signs of estrous and normal luteal function » and Pregnancy success as « correlated with percentage of heifers that reached puberty before or early in breeding season » (Perry, 2012) We also know that the optimal age for first conception in dairy cows is 2 years old ; and that can be apply by the control of the hypothalamic-pituitary axis.

Control of Puberty

Several measurements can be made in order to control puberty; blood hormone concentrations are used to determine the pubertal process and the formation of the CL (Corpus Luteum). At puberty, the cow experiences a decrease in negative feedback of estradiol on GnRH followed by an increase in LH level. That will enhance the follicular growth and result in peripubertal period helped by estradiol secretion. Some recent studies have also showned a correlation between body conditions and normal estrous cycles. Indeed, a normal estrous cycle can be influenced by the age, the BW (Body Weight), the breed and size. An interesting study has proven that Leptin, the hormone for energy expenditure, had a role in regulation of HT/HP axis (Hypothalamus-Pituitary axis). Leptin levels increase at the age of puberty, therefore increasing the total BW. « Heifers can be developed to 50 to 55% of mature body weight before breeding season ». Heifers with lower body weight (53%) were seen being cycling before the start of the breeding season in contrast to heifers with more important body weight (58%). (Perry, 2012). Indeed, we assumed that the best body conditions for heifers to cycle and get pregnant was between 55 to 65% BW. Heifers with 65% BW conceived earlier in general during the breeding season.

Synchronisation of estrous cycle and fertility

An other fabulous discovery was to improve the synchrony of estrous and fertility by controlling both the follicular development and the luteal regression. 2 methods were studied : one based on the control of the luteal function and the follicular waves to improve the pregnancy success and the other related to the hormonal induction of new follicular waves (Perry, 2012). The fisrt method consists to synchronize the follicular waves by « prolonging the lifespan of a dominant follicle » (Perry, 2012). By using Progestin, we can follow the formation of « persistent follicles ». Those modified follicles have an extended lifespan and can increase the concentration of Estradiol (E2) and the LH pulse frequency. AI (Artificial Insemination) applied right after a treatment with Progestin will have consequences in decreasing fertility and alterating the uterine environment. Progesterone in that case will create a « turnover » and therefore make possible the regression and the initiation of new follicular waves (Perry, 2012). The second method focuses on initiating ovulation or atresia in the dominant follicle in a way to generate a new follicular wave. The experiments have shown that Progesterone and Estradiol was the best combination to initiate a new follicular wave after 4 to 5 days after injection. (Perry, 2012). However, GnRH injections were half effective compared the the latest and were also dependent of the stage of the estrous cycle. Further results demonstrated that Progesterone and GnRH injections were negatively related. Indeed, P4 and E2 are important in LH release and the so called “Presynchronisation” that allows a perfect time for the oestrous to optimally respond to GnRH injection and therefore initiating of a new follicular wave. This will have a positive impact on fixed-time AI. (Perry, 2012) That method can be reinforced by the so called “Ovsynch/TAI” (Ovulation-synchronized/Timed Artificial Insemination) protocol. The specificity of this protocol is that heifers can be inseminated at any stage of the oestrous cycle, meaning that no oestrous detection is realised before. A GnRH injection is given randomly during the oestrous cycle and that will initiate a new ovulation or luteinisation of large follicles necessary for the recruitment of new follicular waves. (F. Moreira, 2000)

Fertility

Different factors can play a role in enhancing fertility. One would be the AFC (Antral Follicle Count). This is strongly correlated with pregnancy success: a decrease in AFC will enhance a decrease in P4 and LH receptors. (Perry, 2012). AFC can also influence milk production and longevity because it promotes the optimal development of mammary gland tissue. An optimal AFC is situated between 23 and 24,5 months of age (D. C. Wathes, 2007). Some programs were tested to improve fertility, and that was the case of the so called “in utero” program, based on the relationship between maternal nutrition and some elementary factors such as growth, development and future reproductive performances in offspring. Studies demonstrated that cows restricted with food for the first 110 days of gestation had their calves with fewer AFC compared to the ones supplemented with rich nutrients that participated in increased conception rates. (Perry, 2012). Some other factors are influencing the fertility but will just be listed here: change in energy and protein intake, uterine environment, genetics (age at puberty, heritability…) and stress. Fertility has decreased over the past few years. We recorded a 60% fertility in 1970 in contrast with a 40% fertility in the early 20th century. (D. C. Wathes, 2007). A poor fertility can be the main factor influencing longevity. The actual solution is a better selection in younger ages. There are several investment stages in the production of offspring. The first would be the semen; its cost, quality, fertilisation rates, etc. A study showed that failures in fertilisation were the cause of 10% of the total losses (insemination at inappropriate oestrous time, embryo losses…) (D. C. Wathes, 2007). Based on the same studies we realised that only 40% of the total AI would result in birth.

2) Revue of endocrinological improvements on fertility and production in dairy cows : the role of AMH

First of all, we are going to focus on the Anti-Mullerian Hormone, a hormone similar to growth factor, part of the superfamily of the TGF (Transforming Growth Factor) and which is exclusively secreted by the granulosa cells of healthy follicles. We are going to demonstrate two studies that have proven that AMH is positively related to AFC, ovarian function/ovary size, fertility, birth weight and superovulatory response. AFC, as a reminder, is known as « the average for the maximum number of antral follicles superior to 3 mm in diameter during each of the 2 or 3 consecutive follicular waves during an estrous cycle » (F. Jimenez-Krassel, 2015).

1st experiment : AMH relationship with productive herd life

Initiation of experiment : Heifers with low AMH levels are more likely to have suboptimal fertility, poor reproductive performances leading to a shorter productive herd life and are most of the case removed from the herd. Method of experiment : 281 heifers aged between 11 and 15 months old were tested by single measurement of AMH levels (blood prelevements on coccygeal vein). PGF2α injection was performed previous to the experiment in order to synchronize the estrous cycle. The heifers had to complete 2 lactations and a third one after calving. Different measures of performance and health parameters were also completed here. Both the level of AMH and the size of the ovarian reserve were measured. We applied AI the following day after « standing estrous » until successful pregnancy was diagnosed. To better interpret the results, we used quartiles (Q1, Q2, Q3, Q4) that correspond to the different levels of AMH : Q1 for lowest AMH concentrations and Q4 for the higest ones (F. Jimenez-Krassel, 2015). It is important to notice that, without knowing any result before the end of the experiment (i.e the end of the tree lactations), heifers with decreased or lower productive performances (including milk production, poor reproductive performances…) were intentionally culled. Results : Heifers in the first quartile had shorter productive herd life, reduced survival rate after birth of the first calf, lowest milk production levels, lowest percentage of pregnancy and that quartile contained the highest percentage of culled heifers. However, the upper quartiles (Q2, Q3, Q4) showed longer productive herd life and with higher percentage of remaining cows. (F. Jimenez-Krassel, 2015). Application(s) of experiment: Surprisingly, levels of milk production were not associated with AMH levels in dairy heifers. Ovarian reserve and function are said to be « moderately heritable in dairy cows » (F. Jimenez-Krassel, 2015) ; which can therefore be applied in the actual goals of reproductive herd performance : the AMH concentration and ovarian reserve - related gene selection, also called « AMH-related genetic markers » (F. Jimenez-Krassel, 2015).

Conclusion: By simple determination of AMH levels a diagnosis can be made to anticipate the future productive herd life of dairy cows and their tendency to produce better and in a longer period. “AMH can be a useful phenotypic marker to improve longevity of dairy cows” (F. Jimenez-Krassel, 2015). Furthermore, we can better understand the mechanisms contributing to the relation between AMH, fertility, level of milk production, longevity, etc.

2nd experiment: AMH relationship to super ovulatory response

Initiation of experiment: As defined earlier, AMH gives us information about the ovarian follicular reserve and its main role is to “modulate early follicular growths and inhibit excessive number of follicles from entering growing follicle pool” (A. H. Souza, 2015). This experiment has demonstrated the positive relation between AMH plasma levels and the number of large follicles and CL after both super stimulation and superovulation. Method of experiment: blood samples from approximately 70 cows were taken at 3 different stages of synchronized oestrous cycles (PGF2α injections). Transrectal ultrasonography was used for embryo collection. Results: The same analysis with quartiles was applied. To investigate the results, “animals with 3 or more CL at the time of embryo collection were considered to have responded to the superovulatory treatment” (A. H. Souza, 2015). Q1 seemed to have the lowest superovulatory response compared to the fourth one. Upper quartiles had the following advantages: a greater number of fertilized structures and better transferable and freezable embryos. AMH was then strongly correlated with superovulation response but not to the percentage of fertilized embryos. Additionally, AFC was said to be the primary source of circulating AMH. The results above can demonstrate the “hypothesis that AMH concentrations are predictive of superovulatory response”. Indeed, this method can serve for precise synchronisation of follicular wave before superovulation and therefore evaluating the ovarian reserve. Here, the practical point of view is essential to facilitate new implementations of that technology and to reduce the costs in embryo selection. Another important factor that can be emphasized in that case is the correlation with the body weight (BW). BW can positively alter the circulating AMH concentrations. In opposition to that, “the decrease in AMH may reflect reduced antral follicle numbers in cows with greater BW loss” (A. H. Souza, 2015). Conclusion: The circulating AMH levels are positively correlated with superovulatory response and “embryo production potential of individual cows” (A. H. Souza, 2015). This study shows again a large improvement in embryo-transfer programs concerning dairy cows. Finally, the importance of genetics (AMH-related genetic markers) and those essential parameters studied in both experiments can bring us to a new vision of artificial selection in dairy cattle, strongly influenced by technological advances.

3) Effects of environmental heat stress on reproductive performances in dairy cows

By definition, stress is the “condition where there is undue demand for physical and mental energy due to excessive and adversive environmental factors and cause deformations those are identifiable through physiological desequilibrium” (Abrar Ahmed, 2015). Nowadays, Heat Stress (HS) is considered as a major economical and environmental issue and might lead to economical losses mostly in tropical and some temperate regions, consequences of the climate change that sees its temperatures increasing by 0,2°C in average per decade (Ramendra Das, 2016). The comfort of the dairy cow can be evaluated using the so called term “comfort zone” where energy expenditure of the cow is at its minimum and may be influenced by its age, breed, diet and feed intake. We say that the animal is in HS when it is outside of its comfort zone. The normal body temperature of the cow is between 38,4 and 39,1, its thermoneutral zone ranges from 16 to 25°C. Above an outside temperature of 20-25°C we can say that the cow experiences heat gain which will later result in HS (Ramendra Das, 2016). According to recent studies, we distinguish two types critical temperatures. First the Lower Critical Temperature (LCT) where the “Animal needs to increase its metabolic heat production to maintain its normal body temperature” and second the Upper Critical Temperature (UCT) that makes the animal to elevate its heat production by a rising in body temperature or evaporative heat loss. (Abrar Ahmed, 2015). Those critical temperatures are the consequence of HS and can be manifested by decreasing milk production and reproductive performances.

HS is the cause of several reproductive functions

This table summarises the different effects that HS can cause on reproduction and show how much reproductive functions can be altered by its effect. HS in a general way suppresses endocrinology and immunity of the animal.

Milk production is decreased

Indeed, dairy cows need to have a very efficient and high level metabolism that help them to produce larger quantity of milk every day. When milk production becomes intensive, for example at the lactation peak (4 weeks), heat metabolism is present and heat stress is induced, having strong negative impacts on milk synthesis. Therefore dairy cows are more sensitive and fragile to HS as milk production is getting bigger and bigger. According to some deeper studies, the mechanism is the following: HS influences the activation of “stress response systems in lactating cows” (Ramendra Das, 2016) that will have a negative effect on feed intake. By decreased of food intake, milk production will be lower, such as the DMI (Dry Matter Intake) that can be decreased by 0,85 kg every 1°C rise in temperature, inducing up to 36% decrease in milk production. (Ramendra Das, 2016) To face that issue, several methods were applied but Selection was the most accurate one. By selecting for milk yield, we can attenuate the thermoregulatory range of dairy cows, then “magnifying the depression in fertility” that can be a cause of HS (Abrar Ahmed, 2015).

Solutions and answers to Heat Stress

One of the most known solutions to elevate summer fertility management is the introduction of Artificial Insemination (AI). This program, described earlier, does not need any estrous detection but does not act against the effects of climatic temperatures. Finally the most accurate and simple method to fight HS is the cooling method that can both increase reproductive performances and milk production.