INTRODUCTION
⌅Reproductive efficiency of cattle herds is influenced by several factors, such as the breeding system, the breed used, the management employed on the farm, the sanitary status and nutritional status of the animals (11. Gonçalves LDPN, Prado AJ, Pacheco A, Almeida YEFD, Baruselli PS, Silva WCD, Neves KAL. Reproductive Efficiency of Nelore Cows in Fixed-Time Artificial Insemination Programs with Early Resynchronization. Veterinary Sciences, 2025;12(1):27.,22. Silva FPD, Neves KAL, Correa FR, Silva LK, Batista HR, Silva WC, Caruso NM, Minervino AHH. Follicular Dynamics and Pregnancy Rate in Nelore Heifers Submitted to Fixed-Time Artificial Insemination Protocols (FTAI). Veterinary Sciences. 2022;9(8):377.), as well as the animal welfare associated with heat stress indices (33. Silva WCD, Silva JARD, Camargo-Júnior RNC, Silva ÉBRD, Santos MRPD, Viana RB, Silva AGM, Silva CMG, Lourenço-Júnior JDB. Animal welfare and effects of per-female stress on male and cattle reproduction-A review. Frontiers in Veterinary Science. 2023;10(1):1083469.,44. Silva WCD, Printes OVN, Lima DO, Silva ÉBRD, Santos MRPD, Camargo-Júnior RNC, Barbosa AVC, Silva JAR, Silva AGM, Silva LKX, Araújo CV, Britto EM, Lourenço-Júnior JDB. Evaluation of the Temperature and Humidity Index (THI) to support the implementation of a rearing system for ruminants in the Western Amazon. Frontiers in Veterinary Science. 2023;10(1):1198678.). Despite this variety of factors that interfere with animal reproduction, the use of biotechniques applied to reproduction has shown positive results, being of fundamental importance for the economic growth of national livestock (55. Hallerman EM, Bredlau JP, Camargo LSA, Dagli MLZ, Karembu M, Ngure G, Wray-Cahen D. Towards progressive regulatory approaches for agricultural applications of animal biotechnology. Transgenic Research, 2022;31(2):167-199.,66. Nagy PP, Skidmore JA, Juhasz J. Intensification of camel farming and milk production with special emphasis on animal health, welfare, and the biotechnology of reproduction. Animal Frontiers, 2022;12(4):35-45.,77. Bourdon G, Cadoret V, Charpigny G, Couturier-Tarrade A, Dalbies-Tran R, Flores MJ, Jouneau A. Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies. Veterinary research, 2021;52:1-20.,88. Oliveira BS, Silva KZ, Batista HR, Silva WC, Júnior RNCC. Estudo retrospectivo das taxas de prenhez obtidas com uso de protocolos de inseminação artificial em tempo fixo (IATF) em vacas suplementadas com hormônio liberador de gonadotrofina (GNRH)-Mini Revisão. Brazilian Journal of Development. 2021;7(12):119023-119031.,99. Lazarini IS, Paula-Santos U, Vinhote BP, Silva WC, Júnior RNCC. Prenhez em novilhas Nelore induzidas à puberdade, criadas na Amazônia Oriental Pregnancy in puberty-induced Nelore heifers, breeding in the eastern Amazon. Brazilian Journal of Development. 2021;7(12):119012-119022.,1010. Oliveira VS, Silva WC, Pina-Maia T, Silva, LKX. Is there a correlation between the body condition score and the pregnancy rate of Nelore cows submitted to FTAI in the Eastern Amazon? CES Medicina Veterinaria y Zootecnia. 2022;17(2):8-18.).
The live weight of the animal, the category and the physiological conditions are parameters that vary according to the nutritional, vitamin and mineral conditions of the cattle. In this context, the reproductive phase in bovine females is established as the one with the highest requirement for vitamin and nutritional supplementation and that can affect reproductive efficiency. Thus, the use of vitamins is necessary to maximize reproductive efficiency (1111. D’occhio MJ, Baruselli PS, Campanile G. Influence of nutrition, body condition, and metabolic status on reproduction in female beef cattle: A review. Theriogenology. 2019;125(1):277-284. https://doi.org/10.1016/j.theriogenology.2018.11.010 , 1212. Abera M, Yusuf Mummed Y, Eshetu M, Pilla F, Wondifraw Z. Physiological, biochemical, and growth parameters of Fogera cattle calves to heat stress during different seasons in sub-humid part of Ethiopia. Animals, 2021;11(4):1062.).
The use of vitamins is essential for the growth, reproduction and health of animals and their deficiencies in the body can lead to the emergence of negative symptoms (1313. Izquierdo AC, Reyes AEI, Lang GR, Oaxaca JS, Liera JEG, Mancera EAV, Sánchez RS. Nutrition and Food in the Reproduction of Cattle. European Journal of Agriculture and Food Sciences, 2021;3(3):21-33.). Associated with this, the suplementation of minerals ensures the proper development of the organism and, when administered by injection, it does not suffer interference from dietary antagonists (1414. Priya P, Kumari P, Ballabh J, Ikram M, Jain G, Prasad R, Joshi R. Nutritional Deficiency of Farm Animals: A Review. Journal of Survey in Fisheries Sciences, 2022;349-354.), which causes an increase in these molecules in the animal organism (1515. Arthington JD, Ranches J. Trace mineral nutrition of grazing beef cattle. Animals, 2021;11(10):2767.).
In this context, the use of supplements improves the pregnancy rates of cows in the postpartum period (1616. Younis M, El-Ashker M, El-Diasty M, Youssef MA, El-Khodery S. Oxidative Stress in Transition Dairy Cattle: Current Knowledge and the Potential Impact of Supplementing Organic Trace Elements. Asian Journal of Research in Animal and Veterinary Sciences, 2021;7(1):1-21.,1717. Kotsampasi B, Karatzia MA, Tsiokos D, Chadio, S. Nutritional Strategies to Alleviate Stress and Improve Welfare in Dairy Ruminants. Animals, 2024:14(17):2573.), as well as promoting the reduction of oxidative stress, due to inadequate management (1818. Martorano LG, Vitorino MI, Silva BPPC, Lisboa LS, Sotta ED, Reichardt K. Climate conditions in the eastern amazon: Rainfall variability in Belem and indicative of soil water deficit. African Journal of Agricultural Research. 2017;12(21):1801-1810.). Based on this information, the objective of this study was to evaluate the pregnancy rate of Nelore heifers subjected to artificial insemination combined with mineral and vitamin supplementation.
MATERIAL AND METHODS
⌅Ethical aspects
⌅This study was submitted to and approved by the Ethics Committee on Research and Use of Animals of the Federal University of Western Pará, protocol number No. 0120230238.
Local
⌅The study was conducted on a rural property in the municipality of Uruará, in the state of Pará. The climate is hot and humid (Am4) with annual precipitation between 1900 and 2100 mm, average annual air temperature of 25.6 ºC, and relative humidity ranging from 84 to 86 %. The wettest quarter occurs between the months of February and April and the least rainy between the months of August and October (1818. Martorano LG, Vitorino MI, Silva BPPC, Lisboa LS, Sotta ED, Reichardt K. Climate conditions in the eastern amazon: Rainfall variability in Belem and indicative of soil water deficit. African Journal of Agricultural Research. 2017;12(21):1801-1810.).
Experimental animals
⌅Two hundred forty-two Nelore heifers (Bos taurus indicus), 24 months old, with an average weight of 320±2.5 kg, were used in a semi-intensive system. All heifers were clinically healthy, with no cyclical reproductive changes, evidenced by palpation, with a body condition score of 2.85 to 3.00 (1919. Ayres H, Ferreira RM, Torres-Júnior JRS, Demétrio CGB, Lima CG, Baruselli PS. Validation of body condition score as a predictor of subcutaneous fat in Nelore (Bos indicus) cows. Livestock Science. 2009;123(3):175-179.).
All heifers were raised in an extensive system under the same conditions and received mineral salt (Bellman® 80 ready to the trough) and Panicum maximum cultivar Mombaça grass with access to ad libitum water. They were vaccinated against foot-and-mouth disease, brucellosis and rabies. In addition, they were dewormed subcutaneously with Moxidectin 1mg (Cydectin®), 1ml per 50 kg, 35 days before the start of the experiment.
Experimental design
⌅Heifers were randomly divided into two groups: Control group (n = 121 animals - group I) and Treatment group (n = 121 animals - group II), treated with vitamin and mineral supplementation (Anabolic®. Noxon Animal Health, Rod. Anhanguera, km 296 Distrito Industrial, São Paulo, Brazil) (2020. Anabolic®. https://www.noxon.com.br/anabolic/. Accessed on: January 20, 2025.), at a dose of 10 mL per animal, subcutaneously, at the beginning (D0) of the FTAI protocol. In the control group, the same protocol was carried out without the administration of the vitamin-mineral- supplement. FTAI protocol in both groups was performed by the same applicator.
All heifers were submitted to the FTAI protocol, which consisted of the application of 2ml Estradiol (BE; mention the commercial name and the laboratory) intramuscularly (IM) and the implantation of progesterone (P4, Primer Monodose®, Tecnopec) intravaginally. On day 8, all cows had the implants removed and received 0.5 ml of estradiol cypionate (ECP), 2 ml of Estrom® (Chlorprotenol 24.1 mg) and 1.5 ml of Novormom® (Equine Choreonic Gonadotropin). On day 10, Fixed Time Artificial Insemination (FTAI) was performed (Figure 1).
Ultrasound (Mindray®, DP-10 VET) was used to diagnose pregnancy 30 days after FTAI performance, and the presence of the viable embryo with a heartbeat indicated pregnancy.
Pregnancy rate was calculated using the following formula:
Where: PR = pregnancy rate; NCP = number of pregnant cows; NCT = number of cows treated.
It was assumed that the heifers considered pregnant in this study would also give birth and, consequently, produce calves, which was expressed in the results of the economic rate calculation.
Economic analysis
⌅For the economic analysis, expense composition was made with products and services, considering real market values corresponding to the analysis period (June/2023) (Table 1).
| Product/Procedure | Unit | Quantity | Cost/animal |
|---|---|---|---|
| Protocol | 1 | 1 | 28.50 |
| Supplement | ml | 5 | 4.40 |
| Dose of semen | ml | 1 | 20.00 |
| Labor | Animal | 1 | 25.00 |
| Total | - | - | 77.90 |
Statistical analysis
⌅The individual information of each cow (experimental unit) was filled in spreadsheets for statistical analysis. Statistical analysis for the equality of expected proportions was performed using the chi-square test with continuity corrections at 5 % significance. Data were analyzed using SAS procedures (SAS Inst. Inc., Cary, NC, version 9.3).
RESULTS AND DISCUSSION
⌅Pregnancy rate results were 44,62 % (54/121) for group I and 55,38 % (67/121) for the group to which Anabolic® was applied (Figure 2). There were no statistical differences between the supplemented and control groups (p > 0,2373). Although there were no significant differences between the groups, the use of Anabolic® showed promising results in increasing the pregnancy rate of heifers. There were no studies aimed to evaluate the effect of increasing doses of the use of vitamin-mineral supplements (Anabolic®) on the fertility of dairy cattle. However, it is noted that in the present study, the cows evaluated presented satisfactory results in relation to the pregnancy rate after the administration of Anabolic®. According to Ren et al. (2121. Ren SQ, Wang JW, Chen HY, Xu MQ, Jiang H, Gao Y, et al. Effect of vitamin E on apoptosis and proliferation of bovine granulosa cells using cx43. China Animal Husbandry & Veterinary Medicine. 2016;43(16).), it is important to note that cows injected with the lowest dose of vitamins, despite having lower concentrations of progesterone, were able to sustain similar pregnancy rates, 30 and 45 days after FTAI compared to the other groups evaluated.
It is also noted that the pregnancy rate in the group receiving mineral-vitamin supplements increased by 10,76 %, in relation to the control group. Thus, it was possible to identify that the use of Anabolic® before FTAI tended to increase embryonic survival and pregnancy rate. These data corroborate the studies carried out by Pinheiro et al. (2222. Pinheiro VG, Souza AF, Pegorer MF, Satrapa RA, Ereno RL, Trinca LA, Barros CM. Effects of temporary calf removal and eCG on pregnancy rates for timed insemination in postpartum Nelore cows treated with progesterone. Theriogenology. 2019;71(19):519-524.), who observed an increase in the pregnancy rate only in multiparous cows compared to primiparous cows, when subjected to multivitamin-based FTAI protocols, as it was observed only in heifers in this study. In addition, the aforementioned authors also highlighted that the increase in nutrient demand in primiparous cows associated with the inhibitory effect of sucking may promote a lower frequency of LH pulses in these animals, leading to a lower pregnancy rate.
Binelli et al. and Vasconcelos et al. (2323. Binelli M, Machado R, Bergamaschi MACM, Bertran CM. Manipulation of ovarian and uterine function to increase conception rates in cattle. Journal Animal Reproduction. 2019;6(1):125-134.) state that the efficacy of Anabolic® steroids use varies with the body condition score (BCS), the number of days postpartum, and the association with other management strategies, such as temporary weaning.
Sá Filho et al. (1616. Younis M, El-Ashker M, El-Diasty M, Youssef MA, El-Khodery S. Oxidative Stress in Transition Dairy Cattle: Current Knowledge and the Potential Impact of Supplementing Organic Trace Elements. Asian Journal of Research in Animal and Veterinary Sciences, 2021;7(1):1-21.) and Baruselli et al. (1717. Kotsampasi B, Karatzia MA, Tsiokos D, Chadio, S. Nutritional Strategies to Alleviate Stress and Improve Welfare in Dairy Ruminants. Animals, 2024:14(17):2573.) describe that the use of supplements improves the pregnancy rates of cows in the postpartum period, with a more pronounced effect in those with a lower anestrus or in those that were in anestrus.
Corroborating the above, Rodrigues et al. (1212. Abera M, Yusuf Mummed Y, Eshetu M, Pilla F, Wondifraw Z. Physiological, biochemical, and growth parameters of Fogera cattle calves to heat stress during different seasons in sub-humid part of Ethiopia. Animals, 2021;11(4):1062.) evaluated the use of different vitamins in the fertility of zebu cows and described that the association between them caused greater ovarian activity, as well as better pregnancy rate indexes. Souza et al. (2424. Silva FPD, Neves KAL, Correa FR, Silva LK, Batista HR, Silva WC, Caruso NM, Minervino AHH. Follicular Dynamics and Pregnancy Rate in Nelore Heifers Submitted to Fixed-Time Artificial Insemination Protocols (FTAI). Veterinary Sciences. 2022; 9(8):377.) also found better fertility rates in cows that received vitamin supplementation when comparing the control group and the supplemented group.
With results similar to those of the present study, Gouvêa et al. (2525. Gouvêa VN, Colli MHA, Junior WAG, Motta JCL, Acedo TS, Tamassia LFM, Elliff FM, Mingoti RD, Baruselli PS. The combination of β-carotene and vitamins improve the pregnancy rate at first fixed-time artificial insemination in grazing beef cows. Livestock Science. 2018;217(1):30-36.) administered β-carotene and vitamins to cows 30 days before FTAI, and observed an increase in the pregnancy rate.
The use of minerals and vitamins in cow reproduction is inconsistent, in which it is observed that some studies show an increase in the pregnancy rate (2626. Sales JNS, Pereira RVV, Bicalho RC, Baruselli OS. Effect of injectable copper, selenium, zinc and manganese on the pregnancy rate of crossbred heifers (Bos indicus × Bos taurus) synchronized for timed embryo transfer. Livestock Science. 2011;142(1-3):59-62. https://doi.org/10.1016/j.livsci.2011.06.014.,2727. Mundell LR, Jaeger JR, Waggoner JW, Stevenson JS, Grieger DM, Pacheco LA, Bolte JW, Aubel NA, Eckerle GJ, Macek MJ, Ensley SM, Havenga LJ, Olson KC. Effects of prepartum and postpartum bolus injections of trace minerals on performance of beef cows and calves grazing native range. The Professional Animal Science. 2012;28(1):82-88. https://doi.org/10.15232/S1080-7446(15)30318-1.,2828. Stokes RS, Ralph AR, Mickna AJ, Chapple WP, Schroeder AR, Ireland FA, Shike DW. Effect of an injectable trace mineral at the initiation of a 14 day CIDR protocol on heifer performance and reproduction. Translational Animal Science. 2017;1(4):458-466. https://doi.org/10.2527/tas2017.0050.) and others not (2929. Arthington JD, Martins PGMA, Moriel P, Havenga LJ. Effects of injectable trace minerals at the start of the breeding season on attainment of pregnancy in commercial beef cows. Journal Animal Science. 2014;92(Suppl. 2):723-724.,3030. Willmore CJ, Hall JB, Harrison S, Drewnoski ME. Effect of a trace mineral injection on pregnancy rate of Angus beef heifers when synchronized using the 14- day controlled internal drug-releasing insert-prostaglandin F2α protocol at a commercial feedlot. The Professional Animal Science. 2015;31(6):588-592. https://doi.org/10.15232/pas.2015-01412 ,3131. Gonzalez-Maldonado J, Rangel-Santos R, Rodriguez-de Lara R, Garcia-Pena O. Effect of an injectable trace mineral complex supplementation on development of ovarian structures and serum copper and zinc concentration in over-conditioned Holstein cows. Animal Reproduction Science. 2017;181(1):57-62. https://doi.org/10.1016/j.anireprosci.2017.03.015 ,3232. Stokes RS, Volk MJ, Ireland FA, Gunn PJ, Shike DW. Effect of repeated trace mineral injections on beef heifer development and reproductive performance. Journal Animal Science. 2018;96(9):3943-3954. https://doi.org/10.1093/jas/sky253.). This can be linked to factors such as breed, category, diet provided, reproductive management, and environmental factors.
The economic analysis showed that there was a gross profitability of R$ 38.805,60, considering the calf value of R$ 3.026,00, in which the difference was 13 calves born after FTAI associated with the administration of vitamin-mineral supplements when compared to the non-supplemented group, as observed in Figure 2.
Based on the results found in this study, future research using variable doses, associated with the study of physiological mechanisms and hormone concentrations in cattle, could provide further information capable of pointing to efficient strategies for the use of these products at the time of FTAI. In addition, evaluating other breeds using this protocol could be useful when making efficient choices for producers.
CONCLUSION
⌅Mineral-vitamin supplementation administered before FTAI did not promote a significant increase in the pregnancy rate in Nelore cows with moderate body condition score. However, in the economic bias, the supplementation protocol provided considerable gains to the producer.