Intrinsic and extrinsic factors that influence ovarian environment and efficiency of reproduction in cattle

The emergent concepts on ovary environment, reproductive physiology and the development of pharmacology are constantly supporting the advance of assisted reproduction. Within the last years, the biotechnics related to the synchronization of follicular development and the manipulation of bovine estrus cycle have progressed rapidly and consistently. The combined use of timed-artificial insemination (TAI), superovulation (SOV), ovum pick up (OPU), in vitro embryo production (IVEP) and timed-embryo transfer (TET) has a great potential to improve reproductive outcomes and disseminate selected genetics, diminishing the interval of generations and improving herds genetic gain. However, several factors can potentially affect the efficiency of these procedures. The knowledge of the particularities of the genetic groups, follicular growth manipulation, follicular population predictors, and metabolic and environmental aspects that interfere with ovarian environment and, consequently, oocyte quantity and quality is crucial to optimize the reproductive programs. This review aims to elucidate some factors that affect the ovarian environment and must be well known in order to improve the efficiency of reproduction in cattle.


Introduction
The increasing knowledge on bovine physiology of the estrous cycle enabled the tight control of follicular growing phases using pharmacological strategies, facilitating the reproductive management and supporting the development of biotechnologies of reproduction (Baruselli et al., 2004(Baruselli et al., , 2012;;Lamb et al., 2010).The strategic reproductive management associate with the use of biotechniques of reproduction can be potentially used to disseminate animals with high genetic merit efficiently.Reproductive tools such as timed-artificial insemination (TAI), superovulation (SOV) of selected donor, in vivo embryo production (IVEP), and timed-embryo transfer (TET) had a dramatic growth within the last years, accelerating the selection, multiplication and dissemination of animals with superior genetics e high potential for beef and milk production (Hansen, 2014).
In this context, the present review aims to discuss some key points related to genetics, breed, antral follicle populations, manipulation of ovarian follicular growth, metabolic status (insulin resistance) and environmental factors (heat stress) associated with oocyte and embryo quality.

Influence of genetic group on ovarian characteristics
Several physiological differences between Bos indicus and Bos taurus cattle related to follicular dynamics have been previously reported.The understanding of these differences has been crucial in developing reproductive strategies specific for each genetic group.The Bos indicus cattle are the predominant breeds raised in tropical regions.However, because Bos indicus cattle have subtle differences in their reproductive behavior compared with Bos taurus breeds (Bó et al., 2003;Baruselli et al., 2007;Sartori et al., 2010), one cannot assume that the physiological parameters observed in Bos taurus would be the same as in Bos indicus cattle.
These differences have important practical implications when setting protocols for TAI and TET.The selection of embryo recipients may also be influenced by physiological differences between the genetic groups.For example, because the CL is more difficult to palpate (smaller) in Bos indicus cattle, recipients suitable to receive an embryo may be rejected on CL size evaluation if the particularities of breed are unknown.Previous studies have also shown that the P4 content of the CL and serum P4 concentrations were lower in Bos indicus than in Bos taurus cattle (Segerson et al., 1984).Therefore, conception rates relative to P4 levels in tropical countries, primarily involving Bos indicus recipients on pasture, may be quite different than Bos taurus females maintained in cold-temperate environments with more adequate nutrition.
It has also been reported that IVEP is more efficient in Bos indicus breeds than in Bos taurus breeds (Pontes et al., 2010;Guerreiro et al., 2014).The greater population of antral follicles found in Bos indicus cattle would appear to result in a greater number of suitable oocytes for in vitro culture (Batista et al., 2014).In this context, Bos indicus (Nelore) heifers are reported to have greater number of visualized follicles and to produce greater number of total oocytes per OPU session, cultured COC and blastocyst rates than Bos taurus (Holstein) heifers (Gimenes et al., 2015; Gimenes et al. (2015).

Influence of Anti-Müllerian hormone on ovarian characteristics
The success of SOV and OPU-IVEP is greatly dependent on individual ovarian characteristics that may influence the number and quality of the oocytes that are retrieved (Wise, 1987;Tan and Lu, 1990;Kastrop et al., 1991;Pavlok et al., 1992;Lonergan et al., 1994;Gandolfi et al., 1998).It is known, for example, that the number of antral follicles in the early follicular phase directly correlates with ovarian reserve (Frattarelli et al., 2000).Indeed, the antral follicular population (AFP) directly represents the follicle cohort in the ovaries, which is associated with the number of oocytes retrieved for IVEP.
A large variability of AFP is reported among different cows, however AFP count is highly repeatable within animal (Burns et al., 2005;Ireland et al., 2007), and anti-Müllerian hormone (AMH) can be considered a reliable endocrine marker of ovarian reserve (Ireland et al., 2007(Ireland et al., , 2008;;Monniaux et al., 2012).AMH is a dimeric glycoprotein member of the TGFβ superfamily of growth factors synthesized from granulosa cells of preantral and small antral follicles (growing follicles up to the antral stage or to a diameter of approximately 6 mm) and represents the indirect activity of the follicular pool (Cate et al., 1986;Grootegoed et al., 1994;Durlinger et al., 1999;Weenen et al., 2004).In cattle, circulating AMH concentration can help veterinarians to predict AFP in ovaries (Ireland et al., 2008;Rico et al., 2009;Batista et al., 2014), response to SOV treatments (Rico et al., 2009;Monniaux et al., 2010a, b;Souza et al., 2015), and more recently as a marker to predict IVEP performance of Bos taurus (Guerreiro et al., 2014;Gamarra et al., 2015;Vernunft et al., 2015) and Bos indicus breeds (Guerreiro et al., 2014).
Aiming to determine the relation between AMH and AFP in different genetic groups, our group recently conducted a sequence of studies.In the first study (Baldrighi et al., 2014), despite the high variability in AFP between individuals within each genetic group, the AFP count was greater in Gir (Bos indicus) than in Holstein (Bos taurus) and Murrah (Bubalus bubalis) heifers (P = 0.01; Fig. 1).Similarly, AMH concentration was lower (P < 0.01) for Holstein and Murrah heifers than for Gir heifers.For the three genetic groups studied, a positive relationship between AFP and AMH concentration was detected.Anim.Reprod., v.14, n.1, p.48-60, Jan./Mar.2017 Similarly, in the second study (Batista et al., 2014), the AFP (P < 0.05) and the AMH concentration (P < 0.0001) were higher in Nelore (Bos indicus) than in Holstein (Bos taurus) heifers, and they were correlated.Furthermore, the number of ovarian follicles observed in all evaluation periods (-120, -60 days and 0 days) was correlated with plasma AMH concentrations in both Bos taurus (Holstein) and Bos indicus (Nelore) heifers (Fig. 2).These results suggest that AMH could be a possible long-term endocrine marker of ovarian activity.Therefore, a single blood sample taken at a random stage of the oestrous cycle to measure serum AMH concentration could be considered a reliable phenotypic marker to predict the relative number of follicles, regardless of genetic group.Because genomic information allows producers to identify genetic merit of their animals at premature ages, we have recently investigated the possibility of producing embryos from oocytes of young female calves that were only 2-4 months old.We have found greater plasma AMH concentrations in calves compared to cycling heifers in both genetic groups, Bos indicus and Bos taurus (Fig. 4; Batista et al., 2016).Indeed, it was previously shown that AMH concentrations fall in parallel to the number of ovarian follicles as rodents (Kevenaar et al., 2006) and women (Piltonen et al., 2005) age.Furthermore, a positive correlation was observed between plasma AMH concentration and the number of follicles (P < 0.0001), retrieved COCs (P < 0.0001), COCs cultured (P < 0.0001), cleaved COCs (P < 0.0001 and P = 0.001), and produced blastocysts (P = 0.0003 and P = 0.009) from Bos indicus (Nelore) and Bos taurus (Holstein; Fig. 5) donor calves.However, there was no correlation between circulating AMH levels and cleavage rate (P = 0.24 and P = 0.36), COC culture rate (P = 0.28 and P = 0.07), or blastocyst rate (P = 0.52 and P = 0.08; Batista et al., 2016).0 1 2 3 4 5 6 7 8 9  0 1 2 3 4 5 6 7 8

Metabolic and nutritional factors that influence ovarian characteristics
The nutritional and metabolic status can interfere with follicular growth patterns, secretion of reproductive hormones, and oocyte quality in cattle (Leroy et al., 2008;Ashworth et al., 2009;Batista et al., 2013;Sales et al., 2015;Baruselli et al., 2016;Ferreira et al., 2016a).Thus, metabolic imbalances may cause systemic alterations that can compromise the success of reproductive biotechnologies, such as TAI, SOV and OPU-IVEP (Webb et al., 2004;Adamiak et al., 2005).
Maternal health and nutritional status during gestation have been reported as important factors that interfere on the number of primordial follicles formed during fetal life (Ireland et al., 2011;Evans et al., 2012).In this context, the influence of mother's undernutrition on ovarian status of female offspring was previously investigated (Mossa et al., 2009).Heifers received diets for maintenance or food restriction (0.6 of energetic needs for maintenance) right before conception until 110 days of pregnancy.The AFP and concentration of AMH of the female calves born from undernourished cows were on average 60% lower than from calves born from cows kept under maintenance diets, when they were 7, 18 and 35 weeks of age.Moreover, studies indicate that disruptions on mother's health during gestation may reduce the ovarian follicular reserve.In this basis, cows with high milk somatic cell count, indicating mammary gland infection, gave birth to female calves with almost 50% less AMH concentration than calves born from healthy cows (low somatic cell count; 0.01 ± 0.08 vs. 0.13 ± 0.03 ng/ml; P < 0.05; Ireland et al., 2011;Evans et al., 2012).
On the other hand, the overfeeding can also have negative aspects on reproduction.A common aspect of commercial SOV and OPU-IVEP programs is the use of non-lactating or late lactation cows as oocyte and embryo donors.In these animal categories, the negative effects of overfeeding (excessive energy intake) can compromise in vitro oocyte developmental competence, especially in over-conditioned (high body condition score) females (Adamiak et al., 2005).The mechanisms that mediate these negative effects on oocyte competence may be related to endocrine alterations, such as hyperinsulinemia, peripheral resistance of insulin, and increased glucose and IGF-I, which may interfere with glucose transport in embryo cells and increased apoptosis.
Our research group conducted a study to evaluate the impact of different energy intakes on metabolic profiles and oocyte quality of the nonlactating Gir (Bos indicus) cows submitted to successive OPU sessions (Sales et al., 2015).Diets were formulated to achieve maintenance (M) or 1.7% of maintenance (1.7M) for non-lactating cows.Following 60 days of high energy feeding, cows had reduced in vitro oocyte competence (Fig. 6).Cows fed high-energy diets had greater glucose and insulin concentrations and a greater level of insulin resistance as determined by the glucose tolerance test.Furthermore, cows receiving high-energy diet had lower abundance of transcripts for GLUT1, IGF1R, IGF2R and HSP70.1 genes in oocytes.  .In vitro embryo production in non-lactating cows (n = 14) fed diets to meet 100 or 170% of energy of maintenance and submitted to nine OPU session at 14 day intervals.Adapted from Sales et al. (2015).
Insulin has an important role in cellular metabolism, however, in excess it may interfere with various metabolic and reproductive processes in dairy cows (De Koster and Opsomer, 2013).During early lactation, low circulating insulin concentrations have been associated with impaired fertility by delaying resumption of cyclicity (Gong et al., 2002).Although greater concentrations of insulin are important to restore ovarian cyclicity, it has been shown in heifers that they may also compromise oocyte quality (Adamiak et al., 2005) and, therefore, fertility.In that regard, excessive insulin may reduce oocyte quality in heifers (Adamiak et al., 2005) and IVEP and gene expression linked to cellular metabolism in nonlactating Bos indicus dairy Anim.Reprod., v.14, n.1, p.48-60, Jan./Mar.2017 cows (Sales, 2011).In the latter study, the negative association of excessive energy intake and increased insulin concentrations on IVEP occurred only after 60 days.Thus, prolonged exposure to a high-energy diet was necessary to compromise oocyte quality.On the basis of "Britt's theory" (i.e., folliculogenesis takes at least 60-80 days until an ovulatory follicle stage; Britt, 1992), adverse conditions such as excessive energy balance leading to insulin resistance status can affect folliculogenesis leading to subsequent issues of oocyte competence at the time of ovulation.Therefore, negative effects on oocyte quality and fertility might not be apparent at the onset of insulin resistance.
In another study, early-lactation (110.5 ± 20.8 DIM; n = 70) and late-lactation (425.6 ± 21.0 DIM; n = 67) Holstein cows were subjected to OPU to evaluate oocyte quality and IVEP (Table 2; Ferreira et al., 2011 and reviewed by Baruselli et al., 2016).In addition to increased number of days not pregnant, late-lactation cows had lower milk yield, greater number of previous inseminations and greater BCS than early-lactation cows (Table 2).Regarding OPU-IVEP, late-lactation cows had greater numbers of recovered and viable oocytes compared to early-lactation cows.However, late-lactation cows had decreased rates of blastocyst (P = 0.0005).In addition to fewer embryos produced, late-lactation cows had greater peripheral insulin resistance than earlylactation cows, based on homeostasis model assessment of insulin resistance (HOMA-IR; Table 2; Matthews et al., 1985;Hackbart et al., 2013).The HOMA-IR was calculated according to a formula presented in the previous studies (Matthews et al., 1985;Hackbart et al., 2013): [basal insulin (mIU/ml) x basal glucose (mmol/L)]/22.5.The major purpose of the HOMA-IR is to predict insulin resistance of peripheral tissues based on a single blood sample after an overnight fast.
Moreover, late-lactation cows had lower serum concentrations of both NEFA (P = 0.07) and BHBA (P = 0.01), although there were greater serum concentrations of glucose (P = 0.02) and insulin (P = 0.001) and a greater insulin-glucose ratio (P = 0.001) compared to early-lactation cows.Stage of lactation did not alter other serum metabolites evaluated (Table 2; Ferreira et al., 2016b).Therefore, late-lactation cows from the present study might have been consuming energy in excess of requirements.Supporting the previous data, lactating cows consuming excessive energy intake experienced increased insulin resistance and reduced blastocyst rate compared to cows consuming only adequate amounts of energy (Leiva et al., 2015).Both relative and absolute numbers of copies of mitochondrial DNA (mtDNA) were reduced in oocytes retrieved from late-lactation cows (Table 2; Ferreira et al., 2016a, b), suggesting a disruption of oocyte quality (Ferreira et al., 2016a).In addition, expressions of mitochondrial-related genes (MTCO1, POLG, POLG2, PPARG, TFAM) were increased in late-lactation cows, suggesting the activation of compensatory mechanisms in response to mitochondrial dysfunction (reduced number of copies of mtDNA) aiming to improve the generation of energy (ATP) required during early embryonic development (Ferreira et al., 2016a).Furthermore, there was a greater ratio of BAX/BCL2 in late-lactation cows, indicating an apoptotic phenotype of the oocytes from this category (Ferreira et al., 2016a; Table 2).Overall, on the basis of the available data, we inferred there was a possible association between reduced oocyte quality and insulin resistance status, mostly manifested in late-lactation cows fed a diet with excessive energy.

Environmental factors that influence ovarian characteristics
Mainly in tropical regions, the poor IVEP yields in Bos taurus cattle can be partly attributed to the heat stress (Al-Katanani and Hansen, 2002;Al-Katanani et al., 2002;Ferreira et al., 2011Ferreira et al., , 2016a)).However, previously reports have shown that heat stress also can exert a deleterious effect on ovarian follicular dynamics and oocyte competence in Bos indicus cattle (Torres-Júnior et al., 2008).
A previous seasonal experiment demonstrated that once the pool of ovarian oocytes is damaged by heat stress, two or three estrous cycles are required (after the end of heat stress) to restore the follicular pool and oocyte quality (Roth et al., 2001).However, the study with Bos indicus cows (Torres-Júnior et al., 2008) showed a carry-over effect of heat stress on blastocyst production up to 105 days after the end of the heat stress (Fig. 7).Therefore, it seems that follicles and oocytes are damaged by heat stress during early stages of folliculogenesis, with a delayed deleterious effect on ovarian function.Nevertheless, Bos indicus breeds have been shown to be more resistant to tropical conditions (i.e.elevated temperature and humidity) than breeds that evolved in temperate climates (i.e, Bos taurus, as Holstein).Essentially, the adaptation of certain breeds to elevated heat and humidity is related to their ability to thermoregulate their body temperature (Bennett et al., 1985;Hammond et al., 1996;Gaughan et al., 1999).
Thus, heat stress has a deleterious effect on oocyte quality of both Bos indicus and Bos taurus dairy females, potentially decreasing the results of TAI, SOV and OPU-IVEP procedures.

Conclusion
The success of the application of reproductive biotechniques is closely dependent on individual ovarian characteristics, genetic particularities, nutritional and metabolic status, and environmental factors that may influence the number and quality of the oocytes and embryos.Therefore, factors related to breed, follicular count (AMH), heat stress and nutrition should be considered when applying TAI, SOV, OPU-IVEP and TET in the field.Adequate control of environmental and nutritional conditions should be one of the requisites to be accomplished before implementing any reproduction biotechnology.On the other hand, the knowledge of physiological differences between Bos indicus and Bos taurus cattle is crucial to determine he correct strategies to manipulate follicular wave dynamics for TAI, SOV, OPU-IVEP and TET programs.Additionally, the selection of oocyte and embryo donors with greater follicular population can optimize the efficiency of embryo production techniques.Once these biotechnologies can be efficiently applied on a large scale in the field, significant enhancements in livestock genetic gain can be accomplished with great productivity and economic return for the activity.

Figure 6
Figure6.In vitro embryo production in non-lactating cows (n = 14) fed diets to meet 100 or 170% of energy of maintenance and submitted to nine OPU session at 14 day intervals.Adapted fromSales et al. (2015).

Figure 9 .
Figure 9. Pregnancy per artificial insemination (AI) of Holstein cattle of different categories during summer and winter (heifers = H, high-producing cows in peak lactation = PL and repeat-breeder cows = RB).Adapted from Ferreira et al. (2013).

Table 1 .
).Effect of genetic group on oocyte recovery and quality, and developmental competence of Bos indicus (Nelore) and Bos taurus (Holstein) heifers.

Table 2 .
Ovum pick up, in vitro embryo production and metabolic profile of high production Holstein cows during early or late in lactation.