Effect of the Strategic Supplementation for Producing Embryos of Native Hartón del Valle Heifers

Department of Ruminants, Institute of Animal Science, San José de las Lajas, Mayabeque, La Habana, Cuba Arapaima Fishery and Agroforestry Center, National Learning Service (SENA), Regional Putumayo, Puerto Asís, Putumayo, Colombia Department of Applies Biostatistics, Institute of Animal Science, San José de las Lajas, Mayabeque, La Habana, Cuba Institute of Research on Pastures and Forage, La Habana, Cuba


Introduction
In the technological development of cattle rearing field, there is a search for optimizing the conservation of genetic resources (Prenteci and Anzar, 2011). This is also applied to assisted reproduction. In this regard, embryo production is one of the methods for boosting livestock production in the world mundo (Borge et al., 2019). In native Hartón del Valle cattle, this method is a tool for germplasm conservation, used in Colombia, with institutions such as the Colombian Corporation for Agricultural Research (AGROSAVIA) and private farms (Martínez, 2019). Native breeds are considered to be in danger of extinction. However, variability of embryonic response depends on different factors such as environmental, technical, genetic and nutritional management. The latter takes relevance due to the effect of nutrients on embryonic development (Buerkle, 2007).
In some cases, nutritional balance of donors is inappropriate due to specific conditions of soils (Staal et al., 2020). In the Colombian Amazon, the problem increases because soil is unsuitable for livestock activity, which makes soil-plant-animal relation to be limited for some nutrients nutrientes (Suárez et al., 2018;Mathios Flores et al., 2019;Staal et al., 2020), especially when it is necessary to fulfill the minimum requirements of bovines to obtain favorable results, in terms of oocyte quantity and embryo quality (Calderón et al., 2017). This response is given by the development of antral follicles, mediated by the Follicle-Stimulating Hormone (FSH) action, as one of the important factors that affect embryo production in cattle (Ibtisham et al., 2018). Antral follicle development is correlated with multiple ovulation. A nutritional flushing could increase maturation of primary follicles, due to the increase of Insulin Growth Factor (IGF1) insulina (Larson and White, 2016). These factors increase the sensitivity of granulosa cells and stimulate receptors for Luteinizing Hormone (LH). However, a high amount of insulin in blood compromises embryo viability and quality (Rossetto et al., 2016).
In this context, the Amazonian piedmont includes a wide variety of species, suitable for animal feeding animal (Moniruzzaman and Miyano, 2010;Sotelo et al., 2017, Riascos-Vallejos et al., 2020a. This fact allows to state that there are forage resources of high quality in the Amazonian area, with potential for animal feeding. Supplements based on these species, with proper contents of protein, energy, minerals and degradability, compensate animal requirements because livestock production systems, in this region, base their feeding on native grasses (Riascos-Vallejos et al., 2020b;Cardona-Iglesias et al., 2016).
A diet that meets all nutritional requirements, in different periods, could have a positive effect on embryo production. Therefore, the objective of this research was to evaluate the effect of a strategic supplementation for embryo production of Hartón del Valle heifers.

Animals and Reproductive Diagnosis
For this study, 18 Hartón del Valle heifers were selected, with 34±2 months of age and body weight of 340±20 kg, were selected. Prior to the beginning of experimentation, these animals underwent a general physical examination, including the evaluation of morphology and integrity of their reproductive tract, according to Schneider methodology (Schneider et al., 2012). A portable ultrasound equipment (Econ Control Medical Ref. IMAGO), with a 7.5 MHz linear transducer, was used, to confirm the absence of pregnancy, assess ovarian condition and verify the absence of cysts, adhesions and other pathologies. Furthermore, the absence of fibrosis and malformations was confirmed by passing through the cervix with a catheter.

Feeding
Heifers were fed a diet containing 0.690 kg of crude protein and 79.53 kg of Metabolizable Energy (ME Mjul kg DM 1 ). They received a supplement containing Piptocoma discolor (28, 29), corn, soybean cake, corn bran, molasses, palm oil and mineral premix (Table 1).
A supplement of 2 kg was offered per day in the period of 7:00 bm-9:00 am, then they went to grazing Brachiaria decumbens was used as forage source (Ruíz and Menchaca, 1990). Food balance calculation was performed according to nutritional requirements (NRC, 2001). It was carried out with CALRAC® computer program (version 1.0).

Superovulation Protocol and Embryo Collection
P36/L60 superovulation protocol, developed by (Baruselli et al., 2006;Moraes et al., 2007), was used. This protocol started on day 0 with the placement of an intravaginal device (CIDR® Pfizer Lab), filled with 0.5 g of natural progesterone, plus an Intramuscular (IM) injection of estradiol benzoate at a dose of 2.5 mg (EB; Ric-Be, Syntex SA, Argentina), 600 IU of FSHp and 600 IU of LHp (Pluset® Calier Lab, Spain) in four days, with decreasing applications at 12 h intervals, two doses of cloprostenol sodium (Estrumate® Schering-Plough Lab), each dose of 750 mcg, one in the morning and one in the afternoon on day 6 and the device was removed on day 7. After five days of the beginning of Superovulation (SO), estrus was detected and animals were inseminated at 12, 24 and 36 h post estrus, with a commercial semen of proven fertility (belonging to the same breed). On day 8, 0.25 mg of a synthetic GnRH gonadorelin analog (Fertagyl® Intervet Lab) was applied. On day 15 of treatment, embryo collection was carried out by uterine lavage with a two-way catheter. On this same day, the biological material obtained in filters was evaluated and an intramuscular injection of 750 mcg of cloprostenol sodium (Estrumate® Schering-Plough Lab) was administered (Fig. 1).

Embryo Collection
Embryos were collected seven days after heat was detected. Donors were placed in a restrain cage, where dorsal coccygeal and perivulvar areas were cleaned and disinfected, applying between 5 and 8 mL of 2% lidocaine epidurally. A two-way Foley catheter was vaginally inserted, adjusting it in the horn curvature, to perform uterine infusion-collections. An amount of 1.5 L of phosphate buffer solution was used, PBS with penicillin (100 mg mL 1 ) and streptomycin (100 mg mL 1 ) (ViGro™ Complete Flush Solution®) (Dulbecco and Vogt, 1954).

Embryo Search and Evaluation
On embryo collection day (day 15) and on insemination day (day 8), the number of follicles and corpora lutea were determined by rectal palpation, respectively. Once the lavage of each donor was completed, filter content was placed in a 100100 mm square Petri dish with a gridded background and put in a reflected light stereo microscope with a magnification range from 6.7 to 45 X, for searching embryos. A 35-mm Petri dish was prepared with a maintenance solution (Syngro tm Holding. Bioniche®) to place collected embryos. Selection and classification of collected structures (embryos and oocytes) were performed following the procedure indicated by the International Embryo Transfer Society (IETS) (Stringfellow and Seidel, 1998).
Embryos were classified according to their development stage, on a scale from 1 (one cell stage) to 9 (hatched blastocyst stage) and according to their quality as 1 (excellent), 2 (good), 3 (fair) and 4 (degenerate) (Phillips and Jahnke, 2016). For cryopreservation, morula and blastocyst stage embryos with qualities 1 and 2 were placed in a freezing medium with 10% ethylene glycol and 0.3 M sucrose and placed into French straws (Baruselli et al., 2015).

Economic Analysis
The evaluation was carried out using the partial budgeting technique, which allows to measure direct economic impact of the technological alternative that is intended to be implemented through the relationship between costs and incomes generated by it, compared to the technology that is to be replaced or improved (Mendieta, 1996).

Statistical Analysis
Three groups of animals were used, six replicates each, which were selected from a group of 30 animals. For the variables number of follicles, number of collected embryos and corpora lutea, a non-parametric (Kruskal and Wallis, 1952) analysis of variance was used, according to a completely randomized design. For the comparison of mean ranges, (Conover, 1999) test was applied for p<0.05. For variables TE, NTE and quality of the stages in early morula, morula and blastocysts, a Chi-square comparison proportion analysis (Font et al., 2007) was performed and (Duncan, 1995) test was applied for p<0.05, in the necessary cases. The statistical program InfoStat (Di Rienzo et al., 2012) was used for processing information. Table 2 shows results of the effect of supplementation period. No differences were detected among treatments under evaluation. Biological responses of follicles and corpora lutea were in the range of 6.17 to 8.33 and 5.5 to 7.17, respectively. These results were similar to those reported by (Acosta et al., 2016), who determined the effects of methionine and choline supplementation during prepartum and postpartum periods in embryos before their implantation in Holstein cows.

Number of Follicles and Corpus Luteum
Results regarding number of structures demonstrated that there were no differences among supplementation periods, with means from 2.0 to 3.0 (p = 0.6456) structure treatment 1 . This agrees with reports of (Estrada, 2018), who studied estimated means of structures for seven breeds of native cattle, which ranged between 0.3 and 3.9 viable embryos.
Regarding percentage of extraction, there are no statistical differences among supplementation periods, with means between 38.63 and 42.22 (p = 0.5779). In this sense, response capacity to nutritional programs increases when animal requirements of these programs are fulfilled, depending on soil-plant-animal relationship .

Transferable Embryos
Proportion of transferable embryos, non-transferable embryos and total structures, obtained by the collection in each treatment (Table 3), indicate that TE percentage per treatment, regarding total TE, showed no difference among them, ranging between 28.13 and 40.63%. However, NTE proportion, with respect to total, was lower (p˂0.05) in supplemented treatments, regardless of the period in which it was carried out.
The aforementioned could be favored by supplementation, which provided an energy balance from transfer up to recovery of embryos in cattle, as proposed by (Shorten et al., 2018). In addition, it is stated that cows with higher energy balance have lower embryo losses (Obeidat et al., 2019). Figure 2 shows results of the excellent quality of collected structures. It demonstrates that, when supplementing in both protocol phases, early morulae and blastocyst proportions were increased (p<0.05).
While in morulae, there were no differences among studied treatments. Snider et al. (2019) used a registered nutritional supplement and the results indicated an improvement of embryo quality and a decrease of nontransferable embryos, with a supplementation period of 49 days, before embryo collection.
Results of the current research were consistent with those reported by (Sales et al., 2015), who studied Bos Taurus and Bos indicus donor cows, fed a maintenance diet (maintenance energy and 1.7 higher than maintenance), 21 days before the beginning of superovulation protocol. Bos indicus cows, with a higher energy diet, had better oocyte quality and superior number of viable oocytes. Table 4 shows the economic evaluation. Expenses for superovulation inputs represent between 72.0 and 86.1% of total expenses. Supplementation expenses represented 17% of total expenses in the treatments in which supplementation was use in any of the stages.

Economic Evaluation
Profits increased by 1.53 and 2.05 times when comparing supplementation treatments in both stages and non-supplementation.  Table 4: Economic analysis of embryo production, according to supplementation periods with 20% of Piptocoma discolor  This favored the increase of cost/benefit relationship in 7.72 and 45.09%, after comparing the same treatments.

Discussion
Regarding the number of structures (González et al., 2017), found that the measured ovulatory response in heifers and found an average of 2.0±1.1 embryos per animal. Supplementation favors the response to number of follicles (Occhio et al., 2019), which could be related to changes in dry matter intake or during the supplementation period (Kumar and Laxmi, 2015) and this may propitiate an early embryonic development (Meléndez and Bartolomé, 2017).
These results were consistent with those reported by Molina-Coto et al. (2018). In that study, 300 mL of Propylene Glycol (PPG) were orally provided per day at the beginning of superovulation protocol. It was inferred that, despite PPG was a gluconeogenic supplement and ovaries were glucodependent, PPG supplementation did not affect the amount of collected embryos and follicles (Pérez-Clariget et al., 2017;Ribeiro, 2018). Results of the experiment suggested that other factors unrelated to the effect of supplementation, affected animal response to superovulation and embryo collection treatments (Ruiz et al., 2017).
Another factor was breed, which affects embryonic development during in vitro production, with the use of Swedish red breed as oocyte donors, there were differences with red Holstein regarding percentage of blastocysts (Ballesteros et al., 2017). Results of different studies showed that crosses and Scandinavian red breeds had better fertility than pure Holstein breed (Heins et al., 2006;Aguilar et al., 2018). This performance in native cattle could increase circulating glucose concentrations (Leane et al., 2018), which are positively associated with ovulation rates and in vivo follicular growth and, therefore, with a greater number of transferable embryos (Guanga et al., 2020;Laskowski et al., 2016).
For Chagas et al. (2007) sated that one of the reasons no significant differences were obtained in PPG supplementation, despite a different biological response, was that donors maintained an adequate base nutrition and body condition during the experiment. This limited the response to any supplement. However, improvement was achieved when it was provided to animals with low body condition. These studies were consistent with Hurley and Doane, (1989), who found that superovulated cows with an optimal feeding scheme, supplemented with selenium, showed 100% of fertilized structures, compared to 41% of fertilized egg cells in non-supplemented cows.
Likewise, Abuelo et al. (2015) evaluated the response of Angus heifers to superovulation protocols, which were fed mineral mixtures from organic and inorganic sources for 23 days with respect to a non-supplemented group (C). Although similar amounts of TE were collected per treated heifer, animals of the group of organic sources averaged 0.44 NTE per female treated at the time of collection, which were lower values than those found in the current experiment. Published information indicates that populations of native cattle have, as an average, lower number of transferable embryos than those obtained from other breeds (Mikkola et al., 2020;Robinson et al., 2019).
At the beginning of supplementation, an energy deficit compromises the high levels of urea, which can lead to changes that may produce infertility. This could alter the protein-energy relationship and be the cause of poor embryonic development (Crociati et al., 2017). This could explain the decrease of the number of nontransferable embryos with the use of supplementation (Summers et al., 2018;Kruse et al., 2016).
However, supplementation effects on embryo obtaining were contradictory. Thus, Hackbart et al., (2017) reported that supplementation with PPG reduced total ovulations and fertilization rate. This flexibility in the results of this experiment suggests that response of cows to multiovulation was affected by multiple factors, including paternal effect (Rani et al., 2018;Mikkola and Taponen, 2017;Chinchilla-Vargas et al., 2018). This fact makes difficult to evaluate animal response to a supplement. The supply of 2.0 kg MF 1 of animal supplement 1 day 1 , with an effect on embryonic development, could improve physiological conditions of the animal (Perez et al., 2017;Gallego et al., 2017;Moscoso-Piedra and Cabrera, 2019;Kasimanickam et al., 2020).
Contrary to the findings of this study, for Serrano-Pérez (2020) found that undernourishment did not affect pregnancy in the first week of gestation, as well progesterone levels (Stephenson et al., 2018;Noya et al., 2020). Sartori et al. (2016) showed that Gyr cows had a decrease in blastocyst rate (46.9 Vs. 25.7%) during dry period, in which a diet of 1.7 over the maintenance was offered, for more than 60 days. However, these authors concluded that blastocyst rate was the same in non-lactating Holstein cows before or after being overfed for more than 60 days.
Although oocyte intrinsic quality determines developmental competence, that is, oocyte proportion that becomes blastocysts, environmental conditions have a major impact on their quality (Cuevas et al., 2018;Tahuk et al., 2018). In fact, management conditions influence on the moment of embryonic development, as the first division, beginning of a superior genomic activation and blastocyst quality (Zullo et al., 2016;Gerger et al., 2017;. On the other hand, the number of embryos in this study were low, compared to higher means in other breeds, possibly due to factors such as age and physiological status. Kenny et al. (2018); Krause et al., (2017) reported improvements in oocyte competition, fertilization and formation rates of blastocysts with the aging of animals.
According to Nieddu et al., (2015) it is probable that, with a severe nutritional restriction, with 0.6 of energy requirements for maintenance during the first 100 days of gestation, the number of antral follicles decreases in undernourished mothers.
Results of a 42-day supplementation with treatment 3 were similar to those of Gardinal et al. (2018). These authors evaluated different durations of Whole raw Soybeans (WS) supplementation during prepartum period in embryo quality of transition cows. All groups receiving WS during prepartum period showed the highest value in embryonic quality. As stated by Santos et al. (2016), a gluconeogenic and protein source helps the complete metabolism of glucose that favors reproduction. In turn, blastocyst morphology and its quality are related to glucose consumption. Thus, blastocyst low quality is associated with a lower glucose consumption and the high amount of energy in the diet increases insulin receptors and excesses affect the development of blastocysts (Gobikrushanth et al., 2018).
Studies conducted on humans used C13 fatty acids in hours after culture (when the embryo has 8 cells or more). They improved embryonic quality, which was more important in the final development stages because it stimulates protein kinase C, essential for cell differentiation and growth (Gardner et al., 2011).
Melatonin supplementation reduced apoptosis, increased the proportion of Inner Cell Mass (ICM) to total cells and improved the development of bovine embryos generated via Somatic Cell Nuclear Transfer (SCNT) both in vitro and in vivo. Moreover, global H3 acetyl lysine 9 (H3K9ac) level was significantly elevated in the melatonin-treated SCNT group, indicating that the addition of melatonin may affect nuclear reprogramming, resulting in improved blastocyst quality (Yun-Wei et al., 2018). However, Yun-Wei et al., (2018) stated that flushing only during the period prior to insemination is insufficient and that it should be maintained for at least 21 days after insemination for a positive effect on fertility through ovarian activation (López-Gatius et al., 2017).
Results of the current study agree with Gamarra et al. (2018), who observed a strong positive relationship among circulating levels of Anti-Müllerian Hormone (AMH) and in vivo embryo production after superovulation in cattle. In this regard, nutritional factors, which can be balanced by supplementation, seem to influence plasma AMH levels (Souza et al., 2015;Baruselli et al., 2011), which positively influences on the number of excellent quality embryos in embryo transfer programs.
Values referred in the current study could be related to the amounts of amino acids due to the effect of the microbial protein, according to studies of Chavatte-Palmer et al. (2018). For authors like Stokes et al., (2017), total protein concentration in the follicular fluid was an indicator of the quality of the oocyte, expression, presentation and concentration of different proteins, which varied with follicle size. Furthermore, fluid composition influenced on oocyte quality. Several studies agree that factors associated with good embryonic quality require simple carbohydrates, pyruvate, ATP, microminerals, antioxidants and vitamins (López-Velázquez et al., 2019;Cajas et al., 2019;Torres Osorio et al., 2019;Abdelatty et al., 2018;Agarwal et al., 2018).
Regarding the economic the costs of embryo transfer technique, it was only suitable for registered pure herds or for genofund conservation (Naranjo-Chacón et al., 2016).
Nevertheless the costs of this study are higher than those reported by Castaño (2016) with a conventional embryo transfer protocol and without supplementation with $ 50.85 per embryo. These costs are relatively low, since Bos taurus breed was used, in which the average number of embryos was higher. This increased cost/benefit relationship by achieving a better embryonic response by hormonal methods (Baracaldo Camargo, 2018).
It was recently determined that orally administered propylene glycol to Holstein heifers increased the number of follicles and blastocyst quality of heifers having higher plasma Anti-Müllerian Hormone (AMH) levels. Therefore, measuring AMH before applying FSH programs to cows will allow professionals to increase the number of produced embryos and thus, reduce costs per produced embryo (Sánchez-Castro, 2017).

Conclusion
Supplementation to Hartón del Valle heifers, before and during superovulation, favored the biological response of collected structures, number of follicles and corpora lutea. Similarly, the proportion of transferable embryos, early morulae and quality one blastocytes increased, in addition to achieving a higher cost/benefit relationship.

Funding Information
This research had the financing of 80% of the resources of the National Learning Service SENA; with SENNOVA projects presented by innovation and research lines. Call validity 2018.

Author Contributions
All authors equally contributed in this work.

Conflict of Interest
The authors have no personal financial or nonfinancial competing interest in the product.

Data Availability Statement
Datasets generated for this study are available on request to the corresponding author.

Contribution to Field Declaration
In many species, local genetic resources are indiscriminately crossed with foreign breeds, leading to a reduction or extinction of locally adapted breeds that are promising to adjust to climate change. Native animals demonstrate their ability to survive despite very precarious and inhospitable conditions, such as those found at the time of their introduction by conquistadors.
In addition, it is demonstrated that small existing native groups of animals have a high diversity degree, which indicates a great genetic potential for selection and crossing to produce crossbred animals for their inclusion in the milk and meat production chain. The conservation of genetic resources of native breeds, through embryo collection and diet improvement of supplementation with forages from the Amazonian piedmont, is theoretically important for having updated information regarding the use of local resources for bovine feeding and ovarian activation, in order to increase the production of quality embryos and improve gestation rates.