Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 27/05/2025 Aceptado: 02/08/2025 Publicado: 10/09/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 5 Revista Cienfica, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35709 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Follicular populaon, quanty and quality of cumulus-oocyte complexes of cows grazing in three seasons of the year in the Mexican tropics Población folicular, candad y calidad de complejos cúmulo-ovocitos de vacas en pastoreo en tres estaciones del año en el trópico mexicano Bulmaro Méndez-Argüello 1 , Israel Martínez-Cruz 1 , Cristoforo Mateo Gusmán-Arcos 2 , Froylan Rosales-Martínez 1 *, Rubén Monroy Hernández 1 , Jesús Benjamín Ponce-Noguez 1 , Juan Carlos, Torres-Ramírez 1 ¹Universidad Autónoma de Chiapas, Facultad Maya de Estudios Agropecuarios, Catazajá, Chiapas, México. ²Colegio de postgraduados, Campus Campeche, Champotón, Campeche, México. *Corresponding author: rosales.froylan@unach.mx Tel: 2294201144 ABSTRACT The high temperatures and relave humidity of recent years have cause heat stress in cale, which mainly affects animals in grazing systems. The objecve of this research was to determine the follicular populaon, quanty and quality of cumulus- oocyte complexes in grazing cows during three seasons of the year in the northern regions of Chiapas, Mexico. The study was conducted in three climac seasons: fresh-humid, hot-dry, and hot-humid. A total of 416 ovaries were used (FH=225, HD=100, and HH=91), from crossbred cows (Bos taurus x Bos indicus) from grazing systems. The number of corpus luteum, follicular populaon, and the quanty and quality of cumulus-oocyte complexes, which were also classified as viable and non- viable, were analyzed. The study variables were analyzed using the generalized linear models. No stascal difference was observed, by ovary, for corpora lutea (P ≥ 0.05); with means of 0.35 ± 0.04 (27.3%), 0.46 ± 0.06 (35.9%), and 0.47 ± 0.07 (36.8%) for the fresh-humid, hot-dry, and hot-humid seasons. The season significantly affected medium follicles (P ≤ 0.003), small follicles (P ≤ 0.001), and total follicles (P ≤ 0.001), but not large follicles (P ≥ 0.05). A season effect was observed for all COCs variables (P ≥ 0.05), as well as for the cumulus-oocyte complexes viable and non-viable (P ≤ 0.002). According to the observed results, the highest ferlity of crossbred bovine females occurs during the fresh-humid season, which can be used to schedule breeding and implement reproducve biotechnologies. Key words: Climate change; tropical climate; heat stress; ferlity; reproducon RESUMEN Las altas temperaturas y humedades relavas de los úlmos años, provocan estrés calórico en los bovinos, que afectan mayormente a los animales de sistemas de pastoreo. El objevo de esta invesgación fue determinar la población folicular, candad y calidad de complejos cúmulo-ovocitos de vacas en pastoreo en tres estaciones del año, en la región norte de Chiapas, México. El estudio se realizó en tres estaciones climatológicas, fresca-húmeda, caliente-seca y caliente-húmeda. Se ulizó un total de 416 ovarios (FH=225, HD=100 y HH= 91), provenientes de vacas meszas (Bos taurus x Bos indicus) de sistemas de pastoreo. Se analizó el número de cuerpos lúteos, la población folicular, la candad y calidad de complejos cúmulo-ovocitos, que además se clasificaron en viables y no viables. Las variables de estudio fueron analizadas con el procedimiento de modelos generalizados. No se observó diferencia estadísca, por ovario, para cuerpos lúteos (P ≥ 0,05), las medias fueron de 0,35 ± 0,04 (27,3 %), 0,46 ± 0,06 (35,9 %) y 0,47 ± 0,07 (36,8 %) para las estaciones fresca- húmeda, caliente-seca y caliente-húmeda. La estación afectó significavamente folículos medianos (´P ≤ 0,003), folículos pequeños (P ≤ 0,001) y total de folículos (P ≤ 0,001), pero no folículos grandes (P ≥ 0,05). Se observó efecto de estación en todas las variables de complejos cúmulo-ovocitos (P ≥ 0,05), así como en los complejos cúmulo-ovocitos viable y no viable (P ≤ 0,002). De acuerdo con los resultados observados, la mayor ferlidad de las hembras bovinas meszas se presenta en la estación fresca-húmeda, que puede ser ulizada para programar empadres e implementar biotecnologías reproducvas. Palabras clave: Cambio climáco; clima tropical; estrés calórico; ferlidad; reproducción

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION In the tropical warm climate regions of America, cale ranching is characterized by its producon based on extensive grazing [1]. In these regions, animals are exposed to abrupt environmental changes, which somemes cause stress that affect their ferlity [2]. In recent years, climate elements have changed, partly aributed to greenhouse effects, and the tropical regions have been the most affected [3]. Precipitaon is concentrated more abundantly over short periods, and high radiaon causes prolonged droughts [4]. It has been observed that, in cale, high temperatures and relave humidity cause heat stress in both females and males, compromising their ferlity [5]. Although cale are homeothermic animals that regulate their body temperature to maintain their funcons, heat stress can cause a physiological imbalance, increasing body, vaginal, uterine, and ovarian temperatures in females [6 , 7 , 8 , 9]. In the ovary, heat stress reduces the follicular populaon and affects the development of the preovulatory follicle, which requires an internal temperature up to 1.5 o C lower than in the surrounding ovarian ssues for proper funconing [10]. Peralta-Torres et al. [11], observed in Brahman heifers and their crosses in Mexico, maintained on pasture, the effect of the season of the year on the follicular populaon and the maximum size of the follicles, as well as on their cyclicity. In cumulus-oocyte complexes (COCs), heat stress affects the communicaon between cumulus cells and the oocyte [12], which is essenal for ensuring gestaon in the female [13]. Furthermore, heat stress causes damage to the oocyte´s DNA and to the zona pellucida [14]. In the northern region of Chiapas, located in the southeast of Mexico, the changing climate of the recent years, groups weather seasons with their own characteriscs, where temperature and humidity are high, leading to inferlity issues in cale [2],[15]. Therefore, the objecve of this study was to determine the follicular populaon, quanty and quality of COCs of grazing cows in three seasons of the year in the northern region of Chiapas, Mexico. MATERIALS AND METHODS Characteriscs of the seasons of the year The study was conducted in three climac seasons: fresh- humid (FH, November-February), hot-dry (HD, March-May) and hot-humid (HH, June-October), in Palenque, Chiapas Mexico. The climate of the region corresponds to Af, warm-humid, with rainfall throughout the year [16]. During the enre study period, daily informaon was collected on maximum (Tmax), average (Tmed) and minimum temperature (Tmin), relave humidity (RH) and precipitaon PP), using a weather staon (Vantage VUE ® , Model: 6250, USA) located at the study site. Recovery of ovaries and cumulus-oocyte complexes Ovaries were collected from cows (Bos taurus x Bos indicus) slaughtered at the municipal slaughterhouse in Palenque, Chiapas. A total of 416 ovaries were used (FH=225, HD=100 and HH=91), from crossbreed females Bos taurus x Bos indicus, desned for slaughter. The period from the collecon of the ovaries to their transport to the laboratory was less than half an hour (h). The ovaries were transported in a thermo with one liter of 9% saline soluon (PISA ® , Lab. PISA, S.A de C.V, Mexico), supplemented with 10 µg mL -1 of penicillin-streptomycin (Estreptopen ® , Lapisa ® , Salud Animal, Mexico), at 36 o C. Upon arrival at the laboratory, the ovaries were placed in a water bath (LW Scienfic ® , Model: WBL-10LC-SSD1, Taiwan) and washed with the previously described soluon. The presence of corpus luteum (CL) was visually verified; subsequently, the total number of follicles (5,393) was determined and classified according to their size as small (≤ 4 mm), medium (4.1 to 8 mm) and large (≥8.1 mm) with the help of a veterinary ultrasound equipped with a linear transducer at 7 MHz (Draminski ® , iScan2, Poland) [11]. The COCs (1,798) were recovered by making two incisions in the follicles in the shape of a cross; the follicular fluid was sedimented in sterilized Falcon ® tubes of 15 mL (CTR ® Scienfic, USA) for 15 min at 36 o C. Subsequently, the supernatant was removed and the sediment containing the COCs was placed in 15 mL of Phosphate Buffered Saline (PBS; Sigma Aldrich ® , Germany) at 36 o C. The search and classificaon were carried out using a stereomicroscope (Velab TM , Model: VE-S5C, USA). The PBS containing the COCs was placed in a 100 x 15 mL Petri dish (Falcon ® , CTR ® Scienfic, USA), on a heat plate at 36 o C (TED, Model: MA-3322, USA), for their search and classificaon. Classificaon of cumulus-oocyte complexes The COCs were classified according to their quality into four classes: one (those with more than three compact layers of cumulus cells), two (those with a paral layer of cumulus cells and/or slightly expanded cumulus), three (those with cytoplasm and dark spots, expanded cumulus, indicave of follicular atresia), and four (naked oocytes, without any layer of cumulus cells and that did not necessarily have dark spots and cytoplasm) [17]. Addionally, once classified into classes, the COCs were divided into viable (classes 1 and 2) and non-viable (classes 3 and 4) [18]. Response variables The number of CL was recorded through visual observaon in each ovary, before starng the collecon of COCs; the follicular variables were classified according to what was described by Peralta-Torres et al. [11] into large follicles (FOLG) greater than 8.1 mm, medium (FOLM) from 4.1 to 8 mm, small (FOLP) less than 4 mm, total follicles (TFOL) including FOLG, FOLM and FOLP; the cumulus-oocyte complexes were classified according to what was described by Aguila et al. [17], class one (COCs 1), class two (COCs 2), class three (COCs 3), and class four (COCs 4); and viable (VIAB) and non-viable (UNVIAB) COCs according to what was described by [18]. Stascal analysis The climac characteriscs by season were determined using descripve stascs. For follicular variables and COCs, a linear model was used, considering the fixed effect of the season (FH, HD y HH). The study variables were analyzed using the generalized models (GENMOD) procedure of SAS [19]. A Poisson distribuon was used for the errors with a logisc link funcon. 2 of 5

Ferlity of grazing cows in three seasons of the year / Méndez-Argüello et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico RESULTS AND DISCUSSION The results of this study show that, currently, the precipitaon in the dry season is almost 75% less than in the rainy season and almost 50% less than in the wet season, which leads to an increase in Tmax. The climac characteriscs by season are shown in TABLE I. TABLE I Daily climatological variables (least square means ± standard error) from three seasons of the year, in the northern region of Chiapas Climatological variables Seasons PP (mm d -1 ) Tmax ( o C) Tmed ( o C) Tmin ( o C) RH (%) FH 3.6 ± 1.7 27.4 ± 2.9 21.7 ± 0.7 18.4 ± 1.4 97.5 ± 0.4 HD 1.7 ± 1.5 36.4 ± 4.9 26.3 ± 1.9 23.6 ± 3.5 86.7 ± 5.2 HH 6.7 ± 1.6 34.1 ± 1.2 25.5 ± 0.9 23.7 ± 0.6 94.5 ± 2.8 FH = Fresh-humid; HD = Hot-dry; HH = Hot-humid; PP = Precipitaon; Tmax = Maximum temperature; Tmed = Average temperature; Tmin = minimum temperature; RH = relave humidity. Although the northern region of the state of Chiapas has been classified within the warm humid climates with rain throughout the year [16], in recent years, this phenomenon has been observed: the rainy seasons are becoming shorter and tropical regions have been the most affected [20]. On the other hand, the highest maximum temperatures occurred in the dry season, in the wet and rainy seasons, the values were observed above 90%. This because when the temperature decreases, the relave humidity increases, as colder air has less capacity to retain water vapor. No stascal difference was observed, by ovary, for CL (p ≥ 0.05), with means of 0.35 ± 0.04 (27.3 %), 0.46 ± 0.06 (35.9 %) and 0.47 ± 0.07 (36.8 %) for the FH, HD and HH seasons, respecvely. The presence of a funconal CL in non-pregnant female cale is indicave of their reproducve status since a CL of at least 20 mm in diameter indicates that the females is cycling [21],[22]. In research conducted by Peralta-Torres et al [11] with Brahman and F1 heifers, a higher number of CL was observed in the HH season and although no stascal difference was observed in this research, a slight increase in HH was observed. The season significantly affected FOLM (P ≤ 0.003), FOLP (P ≤ 0.001) y TFOL (P ≤ 0.001) (TABLE II), but not FOLG (P ≥ 0.05). TABLE II Follicular populaon, by ovary (least square means ± standard error), of Bos taurus x Bos indicus cows in three seasons of the year in the northern region of Chiapas Follicular populaon Seasons Large follicles (≥ 8.1 mm) Medium follicles (4.1 a 8 mm) Small follicles (≤ 4 mm) Total follicles FH 0.70 ± 0.06 a 1.41 ± 0.08 a 9.67 ± 0.23 a 11.78 ± 0.25 a HD 0.64 ± 0.08 a 0.92 ± 0.09 b 14.52 ± 0.38 b 16.08 ± 0.40 b HH 0.55 ± 0.08 a 0.86 ± 0.09 b 10.86 ± 0.34 c 12.27 ± 0.37 a a, b. c Different literal in the same column, indicates stascal difference (p ≤ 0.05) between seasons. FH = Fresh-humid; HD = Hot-dry; HH = Hot-humid In this research, although without stascal difference, a greater number of FOLG was observed in FH; these follicles, due to their size, have a higher probability of becoming ovulatory, as they are beer able to take advantage of low concentraons of FSH and the increase in LH. Addionally, the secreon of inhibin B during the follicular phase, produced by the granulosa cells of the developing follicles, decreases the amount of FSH, which helps in the selecon of a single dominant follicle, keeping the remaining follicles in a subordinate status [23]. On the other hand, the greater number of FOLP was observed in HD; these follicles, being very small, have a higher probability of undergoing atresia and not connuing their development due to the dominance of the preovulatory follicle [24]. The results observed in Table 2, may be due to the presence of Tmax above 36 o C in HD, as it has been reported that for each point an increase in temperature and humidity index leads to a decrease of 0.1 mm in follicle size on the day of estrus [25]. In a study conducted by Peralta-Torres et al. [26], using ovaries from Bos indicus and F1 (Bos indicus x Bos taurus) cows from Yucatan, Mexico, they observed 8.10 ± 0.20, 1.82 ± 0.08 and 0.53 ± 0.03 FOLP, FOLM and FOLG, respecvely, in the FH season and 6.81 ± 0.20, 1.49 ± 0.08 and 0.50 ± 0.03, FOLP, FOLM and FOLG, in the HD season; these results are lower than those observed in this research (TABLE II). An effect of season was observed in all COCs variables (p ≥ 0.05) (TABLE III). In this study, similar values of COCs 1 were observed in FH and HD with more than 60% compared to HH. COCs 2 were higher in HD, although likewise, COCs 3 and 4 were greater in the same season. TABLE III Quality of cumulus-oocyte complexes by ovary (least square means ± standard error) of Bos taurus x Bos indicus cows in three seasons of the year in the northern region of Chiapas Cumulus-oocyte complexes Seasons Quality 1 Quality 2 Quality 3 Quality 4 Totals COCs FH 1.55 ± 0.09 a 1.04 ± 0.07 a 1.00 ± 0.07 a 0.12 ± 0.02 a 3.73 ± 0.14 a HD 1.83 ± 0.13 a 1.92 ± 0.14 b 2.64 ± 0.16 b 0.29 ± 0.05 b 6.68 ± 0.26 b HH 0.66 ± 0.08 b 1.09 ± 0.11 a 1.79 ± 0.14 c 0.49 ± 0.07 c 4.04 ± 0.21 a a, b,c . Different literal in the same column, indicates stascal difference (p ≤ 0.05) between seasons. COCs = Cumulus-oocyte complexes. FH = Fresh-humid; HD = Hot-dry; HH = Hot- humid The quality of the COCs is a relevant aspect when evaluang the ferlity of the female bovine, as it has been observed that COCs 1 and COCs 2 have a higher probability of being ferlized [17]. A higher amount of COCs 3 and 4 may be aributed to the high temperatures present in HD, which cause damage to the cumulus cells and the oocyte [27]. There are many factors that affect the quality of COCs, In vitro studies, those collected in hot seasons have shown a lower capacity for development to reach the blastocyst stage [28], as high temperatures alter the expansion of cumulus cells and cytoskeletal components, which leads to increased cellular apoptosis [29]. 3 of 5

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The effect of the season was observed in VIAB COCs (P ≤ 0.002) and UNVIAB COCs (P ≤ 0.002) (FIG. 1). Out of the total COCs collected in the three seasons, 58.8% belonged to the VIAB and only 41.2% to the UNVIAB. The VIAB COCs were 36.5% more in FH compared to HH, which had the lowest count. The highest percentage of UNVIAB COCs was observed in HD, with 11% more compared to the lowest, HH. FIGURE 1. Cumulus-oocyte complexes (%; viable and non-viable) of Bos taurus x Bos indicus cows in three seasons of the year in the northern region of Chiapas In studies conducted in vitro, higher percentages of ferlizaon, embryonic division, and embryos reaching the blastocyst stage have been obtained when using COCs VIAB [17], as it is essenal that cumulus cells and the oocyte are undamaged for proper communicaon [13]. In this study, the highest amount of VIAB was observed in FH and the lowest in HH, which may be aributed to the differences in temperature and humidity present in each season. In in vivo studies, Rosales- Marnez et al. [2 ,[15] with Tropical Milking criollo cows, observed higher pregnancy rates in the FH season, with a decrease from 90 to less than 25% as temperature and relave humidity increased from 20 to 35 o C and 60 to 90%, respecvely. The highest percentage of UNVIAB COCs observed in HD may be due to the stress that female bovines experience when subjected to high temperatures during the dry season, since as body, vaginal [30] and ovarian temperature also increase, altering hormonal secreon and decreasing the competence of the oocytes [31]. Research has been conducted to evaluate the effect of heat stress on the viability of COCs, and it has been observed that the season has a strong impact on their quality [11]. VIAB COCs can be used for in vitro ferlizaon programs and are one of the main indicators of female ferlity. CONCLUSION According to the results observed in this research, the percentage of luteal bodies is similar in the fresh-humid and hot-dry seasons. However, the follicular populaon, with large and medium follicles that have a higher probability of ovulang, was observed in the fresh-humid season. On the other hand, the lowest number of cumulus-oocyte complexes of quality three and four was observed in the fresh-humid season, as well as the highest number of viable ones. Therefore, under the climac condions of the northern region of Chiapas, the highest ferlity of crossbred bovine females occurs in the fresh-humid season, which can be used to Schedule mang and implement reproducve biotechnologies. Conflict of interest The authors declare that they have no conflicts of interests. BIBLIOGRAPHIC REFERENCES [1] Figueroa D, Galicia L, Suárez-Lastra, M. Lan American cale ranching sustainability debate: An approach to social-ecological systems and spaal-temporal scales. Sustainability [Internet]. 2022; 14(14):8924. doi: hps:// doi.org/gv5jsx [2] Rosales-Marnez F, Rosendo-Ponce A, Cortez-Romero C, Gallegos-Sánchez J, Cuca-García JM, Becerril-Pérez CM. Relaon of the maximum temperature and relave humidity close to the inseminaon with the tropical milking criollo heifer´s gestaon in three seasons. Trop. Anim. Health Prod. [Internet]. 2021; 53(1):27. doi: hps://doi.org/p42n [3] Byrme MP, Pendergrass AG, Rapp AD, Wodzicki KR. Response of the intertropical convergence zone to climate change:locaon, width, and strength. Curr. Clim. Change Rep. [Internet]; 2018; 4:355-370. doi: hps:// doi.org/gj74kd [4] Lyra A, Imbach P, Rodríguez D, Chan CS, Georgiou S, Garofolo L. Projecons of climate change impacts on central America tropical rainforest. Clim. Change. [Internet]. 2017; 141:93-105. doi: hps://doi.org/f9xtwd [5] Silva WC, Silva JAR, Camargo-Junior RNC, Silva EBR, Santos MRP, Viana RB, Silva AGM, Silva CMG, Lourenço-Júnior JB. Animal welfare and effects of per-females stress on male and cale reproducon – A review. Front. Vet. Sci. [Internet]. 2023; 10:1083469. doi: hps://doi.org/p42p [6] Chavez MI, García JE, Vélis FG, Gaytán LR, de Sanago A, Mellado M. Effects of in utero heat stress on subsequent reproducon performance of first-calf Holstein heifers. Span. J. Agric. Res. [Internet]. 2020; 18(2):e0404. doi: hps://doi.org/p42q [7] Tian H, Liu J, Chen X, Li S, Li X, Mengal K, Lu Y, Wang D. Effects of ambient temperature and humidity on body temperature and acvity of heifers, and a novel idea of heat stress monitoring. Anim. Prod. Sci. [Internet]. 2021; 61(15):1584-1591. doi: hps://doi.org/p42r [8] Togoe D, Minca NA. The impact of heat stress on the physiological, producve and reproducve status of dairy cows. Agriculture. [Internet]. 2024; 14(8):1241. doi: hps://doi.org/p42s 4 of 5

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