Published knowledge about cat breeders' habits regarding breeding management and contraception is limited, but vital for efficient veterinary support of catteries. With an online questionnaire, we surveyed breeders’ origin, breed, number of breeding queens/tomcats in the cattery, age at first estrus, use of contraceptives and ovulation-inducing drugs or treatment in queens [progestin pills; proligestone injection; hCG; GnRH agonists as injection or deslorelin implants (DSRI); melatonin; vaginal stimulation], as well as the use of contraceptives in tomcats [progestins pills; proligestone injection; DSRI; melatonin]. The participating 404 breeders of 46 cat breeds originated from 24 countries (German (201, 49.8 %) and Bengal breeders (73, 15.8 %) overrepresented) with the majority having ≥10 years of experience (n = 195, 48.3 %). In average, 3–4 intact adult queens (n = 185, 45.8 %) and one intact tomcat (n = 149, 36.9 %) were kept per cattery. Breeders answered that 50.6 % (n = 896) of their queens were 7–10 months old at first heat, long-haired breeds significantly older than short-haired (p < 0.0001). Progestin pills (n = 235, 47.4 %) were most commonly administered in queens, followed by DSRI (n = 53, 10.7 %). Regarding tomcats, the slight majority of breeders (n = 229, 53.3 %) did not use contraceptives, next common were DSRI (n = 141, 32.8 %). Within the study population, contraceptives were most popular in (Northern) Europe, while least common in Northern America. Despite its limitations, this large worldwide study provides relevant insights into reproductive and contraceptive management of catteries. Limited access to contraceptives and dissatisfaction with local veterinary support as commented by several participants suggest the establishment of telephone consultation services and breeder seminars by specialized veterinarians.
{"title":"Insights into breeding management and contraception in catteries – What we learned from an online survey","authors":"Ricarda Schaper, Eva-Maria Packeiser, Sandra Goericke-Pesch","doi":"10.1016/j.theriogenology.2025.117385","DOIUrl":"10.1016/j.theriogenology.2025.117385","url":null,"abstract":"<div><div>Published knowledge about cat breeders' habits regarding breeding management and contraception is limited, but vital for efficient veterinary support of catteries. With an online questionnaire, we surveyed breeders’ origin, breed, number of breeding queens/tomcats in the cattery, age at first estrus, use of contraceptives and ovulation-inducing drugs or treatment in queens [progestin pills; proligestone injection; hCG; GnRH agonists as injection or deslorelin implants (DSRI); melatonin; vaginal stimulation], as well as the use of contraceptives in tomcats [progestins pills; proligestone injection; DSRI; melatonin]. The participating 404 breeders of 46 cat breeds originated from 24 countries (German (201, 49.8 %) and Bengal breeders (73, 15.8 %) overrepresented) with the majority having ≥10 years of experience (n = 195, 48.3 %). In average, 3–4 intact adult queens (n = 185, 45.8 %) and one intact tomcat (n = 149, 36.9 %) were kept per cattery. Breeders answered that 50.6 % (n = 896) of their queens were 7–10 months old at first heat, long-haired breeds significantly older than short-haired (p < 0.0001). Progestin pills (n = 235, 47.4 %) were most commonly administered in queens, followed by DSRI (n = 53, 10.7 %). Regarding tomcats, the slight majority of breeders (n = 229, 53.3 %) did not use contraceptives, next common were DSRI (n = 141, 32.8 %). Within the study population, contraceptives were most popular in (Northern) Europe, while least common in Northern America. Despite its limitations, this large worldwide study provides relevant insights into reproductive and contraceptive management of catteries. Limited access to contraceptives and dissatisfaction with local veterinary support as commented by several participants suggest the establishment of telephone consultation services and breeder seminars by specialized veterinarians.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117385"},"PeriodicalIF":2.4,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143609918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-06DOI: 10.1016/j.theriogenology.2025.117376
Dapeng Yang , Ligang Yuan , Jianlin Zeng , Yumei Qi , Long Ma , Haijun Li , Jinhan Lv , Yulu Chen
Yak is a large plateau mammal with low reproduction rate, while cryptorchidism is a major reproductive disorder associated with infertility in highland yaks. To better understand the occurrence of cryptorchidism and its regulatory mechanisms in yaks, we conducted a multi-omic analysis, and screened a total of 4456 differentially expressed genes (DEGs) and 332 differentially expressed proteins (DEPs) between normal and unilateral cryptorchid testes of yaks using high-throughput transcriptome sequencing (RNA-seq) and Tandem Mass Tag (TMT)-based proteomics techniques, with testes from yaks with cryptorchidism as the target. Enrichment analysis revealed that the DEGs were associated with cell growth, sperm motility, immune regulation, and intercellular tight junctions, and were mainly enriched in pathways related to cell differentiation; amino acid, sugar, and lipid metabolism; cell adhesion, and hypoxia tolerance. The results of protein interactions network analyses indicated tight interactions between the differential proteins CCT2, CCT4, CCT5, FZR1, and PSMA8. In conclusion. This expression of these differential genes and proteins dysregulation may lead to the obstruction of the testicular descent process or the abnormal development of the testis, potentially leading to cryptorchidism. The results of this study laid a foundation for the screening of key candidate genes and proteins for cryptorchidism in yak, and also provided a theoretical basis for the research molecular mechanism of reproductive system diseases in yak and plateau animals.
{"title":"Comparative proteomic and transcriptomic analysis of testicular tissue of yaks with or without cryptorchidism","authors":"Dapeng Yang , Ligang Yuan , Jianlin Zeng , Yumei Qi , Long Ma , Haijun Li , Jinhan Lv , Yulu Chen","doi":"10.1016/j.theriogenology.2025.117376","DOIUrl":"10.1016/j.theriogenology.2025.117376","url":null,"abstract":"<div><div>Yak is a large plateau mammal with low reproduction rate, while cryptorchidism is a major reproductive disorder associated with infertility in highland yaks. To better understand the occurrence of cryptorchidism and its regulatory mechanisms in yaks, we conducted a multi-omic analysis, and screened a total of 4456 differentially expressed genes (DEGs) and 332 differentially expressed proteins (DEPs) between normal and unilateral cryptorchid testes of yaks using high-throughput transcriptome sequencing (RNA-seq) and Tandem Mass Tag (TMT)-based proteomics techniques, with testes from yaks with cryptorchidism as the target. Enrichment analysis revealed that the DEGs were associated with cell growth, sperm motility, immune regulation, and intercellular tight junctions, and were mainly enriched in pathways related to cell differentiation; amino acid, sugar, and lipid metabolism; cell adhesion, and hypoxia tolerance. The results of protein interactions network analyses indicated tight interactions between the differential proteins CCT2, CCT4, CCT5, FZR1, and PSMA8. In conclusion. This expression of these differential genes and proteins dysregulation may lead to the obstruction of the testicular descent process or the abnormal development of the testis, potentially leading to cryptorchidism. The results of this study laid a foundation for the screening of key candidate genes and proteins for cryptorchidism in yak, and also provided a theoretical basis for the research molecular mechanism of reproductive system diseases in yak and plateau animals.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117376"},"PeriodicalIF":2.4,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143577866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-05DOI: 10.1016/j.theriogenology.2025.117375
Rafał Ciaputa , Eleonora Brambilla , Stanisław Dzimira , Marcin Nowak , Izabela Janus-Ziółkowska , Aleksandra Piotrowska , Alicja Tomaszek , Małgorzata Kandefer-Gola , Valeria Grieco
Testin (TES), a protein localised in the cytoplasm and belonging to the LIM family of proteins, is part of the cytoskeleton localised along stress fibres and recruited to focal adhesions. It is considered a tumour suppressor protein in humans and decreased TES expression has been shown to increase cell motility and decrease cell-cell contact. In veterinary medicine, TES has only been studied in rat testes and, more recently, also in canine testes. The expression of this protein in testicular tumours is unknown. As the dog has been proposed as an animal model for the study of human testicular tumours, studies on canine TES may provide useful information for both species. Therefore, the purpose of the present study was to demonstrate the expression of TES in the most common types of canine testicular tumours. For this study, paraffin blocks of 166 canine testicular tumours (53 Sertoli cell tumours, 50 Leydig cell tumours, and 63 seminomas) were retrieved from the archive of the Department of Pathology of Wroclaw University of Environmental and Life Sciences. Sections were obtained for complete description of the tumours, confirmation of the histological diagnosis, and immunohistochemical TES antigen location. Sections from 10 normal canine testes were examined as controls. The presence of TES was also demonstrated in fresh tissue from three types of canine testicular tumour. The diagnosis was confirmed in the 166 tumours, and TES was immunohistochemically demonstrated in 98 % of them and in all 10 normal testes. All tumour types had reduced expression of TES, even if not related to the tumour growth pattern or mitotic index, demonstrating that TES expression is reduced in canine as well in human testicular tumours. However, the reduction of TES expression appeared to be moderate, and these features were consistent with the evidence that testicular tumours in dogs are often well-differentiated. A complete understanding of the function of TES in cancer processes requires further investigation, but the relative parallelism between normal and neoplastic cells in well-differentiated, mostly benign testicular tumours provides a good basis for studying TES expression in more aggressive tumours, such as prostatic or urinary tract neoplasms.
{"title":"Immunohistochemical expression of testin protein in testicular tumours in dogs","authors":"Rafał Ciaputa , Eleonora Brambilla , Stanisław Dzimira , Marcin Nowak , Izabela Janus-Ziółkowska , Aleksandra Piotrowska , Alicja Tomaszek , Małgorzata Kandefer-Gola , Valeria Grieco","doi":"10.1016/j.theriogenology.2025.117375","DOIUrl":"10.1016/j.theriogenology.2025.117375","url":null,"abstract":"<div><div>Testin (TES), a protein localised in the cytoplasm and belonging to the LIM family of proteins, is part of the cytoskeleton localised along stress fibres and recruited to focal adhesions. It is considered a tumour suppressor protein in humans and decreased TES expression has been shown to increase cell motility and decrease cell-cell contact. In veterinary medicine, TES has only been studied in rat testes and, more recently, also in canine testes. The expression of this protein in testicular tumours is unknown. As the dog has been proposed as an animal model for the study of human testicular tumours, studies on canine TES may provide useful information for both species. Therefore, the purpose of the present study was to demonstrate the expression of TES in the most common types of canine testicular tumours. For this study, paraffin blocks of 166 canine testicular tumours (53 Sertoli cell tumours, 50 Leydig cell tumours, and 63 seminomas) were retrieved from the archive of the Department of Pathology of Wroclaw University of Environmental and Life Sciences. Sections were obtained for complete description of the tumours, confirmation of the histological diagnosis, and immunohistochemical TES antigen location. Sections from 10 normal canine testes were examined as controls. The presence of TES was also demonstrated in fresh tissue from three types of canine testicular tumour. The diagnosis was confirmed in the 166 tumours, and TES was immunohistochemically demonstrated in 98 % of them and in all 10 normal testes. All tumour types had reduced expression of TES, even if not related to the tumour growth pattern or mitotic index, demonstrating that TES expression is reduced in canine as well in human testicular tumours. However, the reduction of TES expression appeared to be moderate, and these features were consistent with the evidence that testicular tumours in dogs are often well-differentiated. A complete understanding of the function of TES in cancer processes requires further investigation, but the relative parallelism between normal and neoplastic cells in well-differentiated, mostly benign testicular tumours provides a good basis for studying TES expression in more aggressive tumours, such as prostatic or urinary tract neoplasms.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117375"},"PeriodicalIF":2.4,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143629492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Research suggests that assisted reproductive technologies may lead to adverse effects on the offspring. To date, long-term effects from fetal development through adulthood have been scarcely researched. In the present observational study, we aimed to describe growth (body weight, BW; crown-rump length, CRL; and average daily gain, ADG), and growth-related hormone levels (growth hormone, GH; insulin-like growth factor 1, IGF-1; thyroxine, T4; cortisol) of pigs derived from artificial insemination (AI) and from embryos produced in vitro under two different conditions, C-IVP, with Bovine Serum Albumin (BSA), and RF-IVP, with reproductive fluids and BSA, as protein source in culture, from birth to 5 years of age. In vitro-produced animals showed greater BW, CRL and ADG than AI. Additionally, C-IVP animals had greater BW, CRL and ADG than RF-IVP up to 6 months of age. Regarding hormones, GH concentration was greater in RF-IVP compared to AI and C-IVP. In males, IGF-1 levels of RF-IVP were greater than those of AI. Instead, T4 levels of AI males were greater than C-IVP and RF-IVP. Males showed greater IGF-1 and GH than females, the latter only from 1 year old. Instead, females had greater T4 concentration than males. No differences in cortisol were observed between groups or sexes. In conclusion, this study sets reference values of growth and hormone concentrations for adult pigs that are missing in literature. Although the differences observed between the experimental groups were within physiological ranges, there was a greater growth performance in the IVP groups.
{"title":"Growth performance in pigs derived from in vitro produced embryos is enhanced compared to their artificial insemination-derived counterparts from birth to adulthood","authors":"Sonia Heras , Cristina Soriano-Ubeda , Armando Quintero-Moreno , Jon Romero-Aguirregomezcorta , Evelyne Paris-Oller , Joaquin Gadea , Raquel Romar , Sebastian Canovas , Pilar Coy","doi":"10.1016/j.theriogenology.2025.117372","DOIUrl":"10.1016/j.theriogenology.2025.117372","url":null,"abstract":"<div><div>Research suggests that assisted reproductive technologies may lead to adverse effects on the offspring. To date, long-term effects from fetal development through adulthood have been scarcely researched. In the present observational study, we aimed to describe growth (body weight, BW; crown-rump length, CRL; and average daily gain, ADG), and growth-related hormone levels (growth hormone, GH; insulin-like growth factor 1, IGF-1; thyroxine, T4; cortisol) of pigs derived from artificial insemination (AI) and from embryos produced <em>in vitro</em> under two different conditions, C-IVP, with Bovine Serum Albumin (BSA), and RF-IVP, with reproductive fluids and BSA, as protein source in culture, from birth to 5 years of age. <em>In vitro</em>-produced animals showed greater BW, CRL and ADG than AI. Additionally, C-IVP animals had greater BW, CRL and ADG than RF-IVP up to 6 months of age. Regarding hormones, GH concentration was greater in RF-IVP compared to AI and C-IVP. In males, IGF-1 levels of RF-IVP were greater than those of AI. Instead, T4 levels of AI males were greater than C-IVP and RF-IVP. Males showed greater IGF-1 and GH than females, the latter only from 1 year old. Instead, females had greater T4 concentration than males. No differences in cortisol were observed between groups or sexes. In conclusion, this study sets reference values of growth and hormone concentrations for adult pigs that are missing in literature. Although the differences observed between the experimental groups were within physiological ranges, there was a greater growth performance in the IVP groups.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117372"},"PeriodicalIF":2.4,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143577865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-03DOI: 10.1016/j.theriogenology.2025.117373
Camilo Hernández-Avilés, Dickson D. Varner, Luisa Ramírez-Agámez, Juan C. Samper , Charles C. Love
In the current study, we report the effect of different commercially available semen freezing extenders utilized for the “test-freeze” procedure for 13 years (2010–2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility – TM [%]; progressive motility – PM [%]; and curvilinear velocity – VCL [μm/s]), sperm viability (VIAB [%]), and sperm DNA damage (COMPɑt [%]) were compared among semen freezing extenders. Overall, TM, PM, and VIAB were similar for extenders LE, CMLE, and BC (P > 0.05) while higher than for extenders MFR5 or CMMFR5 (P < 0.05). Mean VCL was lower for extender LE than for extender BC (P < 0.05). An effect of semen extenders on mean COMPαt was not observed (P > 0.05). The proportion of ejaculates that yielded four different post-thaw sperm “freezability” indices, based on the average value between post-thaw TM and VIAB (MV; MV ≤ 41 % [LOW]; MV 42–54 % [AVERAGE]; or MV ≥ 55 % [HIGH]), or post-thaw PM: ≥30 %, was calculated. Extenders MFR5 and CMMFR5 yielded a higher proportion of stallions in the LOW category (36 and 37 %, respectively) compared to extenders LE (22 %), CMLE (20 %), or BC (21 %; P < 0.05). For the HIGH and PM ≥ 30 % categories, extenders LE (16 and 65 %), CMLE (15 and 66 %), and BC (15 and 61 %) yielded a higher proportion of stallions than extenders MFR5 (4 and 40 %) or CMMFR5 (2 and 45 %), respectively (P < 0.05). For the AVERAGE category, an effect of semen extenders was not observed (60–63 %; P > 0.05). The relationship between pre-freeze sperm quality parameters (i.e., sperm motility, morphology [Normal], viability, and COMPɑt) and the indices of post-thaw “freezability” (LOW, AVERAGE, HIGH, PM ≥ 30 %) was also determined. The percentages of TMOT and Normal in fresh semen yielded the highest ROC values related to “freezability,” irrespective of the index used: 1) LOW = % Normal – AUC 0.8328 (P < 0.0001); 2) AVERAGE = TMOT – AUC 0.5875 (P = 0.055); 3) HIGH = % TMOT – AUC: 0.8015 (P < 0.0001); 4) PM ≥ 30 % = % Normal – AUC 0.8392 (P < 0.0001). This study provides clinically relevant data regarding the effect of different semen freezing extenders on post-thaw stallion sperm quality and the potential relationship between some sperm quality parameters commonly assessed in practice and post-thaw sperm “freezability” in stallions.
{"title":"Results of the “test-freeze” approach in a commercial program of stallion sperm cryopreservation and the relationship between pre-freeze sperm quality and “freezability.”","authors":"Camilo Hernández-Avilés, Dickson D. Varner, Luisa Ramírez-Agámez, Juan C. Samper , Charles C. Love","doi":"10.1016/j.theriogenology.2025.117373","DOIUrl":"10.1016/j.theriogenology.2025.117373","url":null,"abstract":"<div><div>In the current study, we report the effect of different commercially available semen freezing extenders utilized for the “test-freeze” procedure for 13 years (2010–2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility – TM [%]; progressive motility – PM [%]; and curvilinear velocity – VCL [μm/s]), sperm viability (VIAB [%]), and sperm DNA damage (COMP<sub>ɑt</sub> [%]) were compared among semen freezing extenders. Overall, TM, PM, and VIAB were similar for extenders LE, CMLE, and BC (P > 0.05) while higher than for extenders MFR5 or CMMFR5 (P < 0.05). Mean VCL was lower for extender LE than for extender BC (P < 0.05). An effect of semen extenders on mean COMP<sub>αt</sub> was not observed (P > 0.05). The proportion of ejaculates that yielded four different post-thaw sperm “freezability” indices, based on the average value between post-thaw TM and VIAB (MV; MV ≤ 41 % [LOW]; MV 42–54 % [AVERAGE]; or MV ≥ 55 % [HIGH]), or post-thaw PM: ≥30 %, was calculated. Extenders MFR5 and CMMFR5 yielded a higher proportion of stallions in the LOW category (36 and 37 %, respectively) compared to extenders LE (22 %), CMLE (20 %), or BC (21 %; P < 0.05). For the HIGH and PM ≥ 30 % categories, extenders LE (16 and 65 %), CMLE (15 and 66 %), and BC (15 and 61 %) yielded a higher proportion of stallions than extenders MFR5 (4 and 40 %) or CMMFR5 (2 and 45 %), respectively (P < 0.05). For the AVERAGE category, an effect of semen extenders was not observed (60–63 %; P > 0.05). The relationship between pre-freeze sperm quality parameters (i.e., sperm motility, morphology [Normal], viability, and COMP<sub>ɑt</sub>) and the indices of post-thaw “freezability” (LOW, AVERAGE, HIGH, PM ≥ 30 %) was also determined. The percentages of TMOT and Normal in fresh semen yielded the highest ROC values related to “freezability,” irrespective of the index used: 1) LOW = % Normal – AUC 0.8328 (P < 0.0001); 2) AVERAGE = TMOT – AUC 0.5875 (P = 0.055); 3) HIGH = % TMOT – AUC: 0.8015 (P < 0.0001); 4) PM ≥ 30 % = % Normal – AUC 0.8392 (P < 0.0001). This study provides clinically relevant data regarding the effect of different semen freezing extenders on post-thaw stallion sperm quality and the potential relationship between some sperm quality parameters commonly assessed in practice and post-thaw sperm “freezability” in stallions.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"238 ","pages":"Article 117373"},"PeriodicalIF":2.4,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143549544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-28DOI: 10.1016/j.theriogenology.2025.02.025
Penelope Maria Gugole , Augusta Zannoni , Monica Forni , Eleonora Iacono , Filippo Zambelli , Barbara Merlo
This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10−8 M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10−8 M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At survival rate determination, the degeneration rate was higher in VIT and VIT-Nx compared to F (P < 0.05). The highest maturation rate was observed in VIT-Nx. A significant reduction in ROS levels was observed in H compared to F (P < 0.05). ROS levels were similar between F and VIT, while the Nx supplementation tended to increase them (VIT-Nx vs F: P = 0.053; VIT-Nx vs VIT: P = 0.069). Conversely, in oocytes vitrified after overnight holding, vitrification induced an increase in ROS levels (H vs VIT: P < 0.05), which was not observed in H-VIT-Nx. GSH intracellular levels showed significant differences only in held oocytes, with higher GH levels in H compared to H-VIT and H-VIT-Nx (P < 0.05). All treatments induced an increase in HMMP levels compared to F (P < 0.05). In H oocytes, mitochondria were distributed throughout the entire oolemma (TOMM20) and active mitochondria (D-LAT) were detected in the outermost region. Incontrast, in H-VIT-Nx, potentially active mitochondria were spread throughout the cytoplasm. AnnexinV/PI staining revealed that the percentage of viable oocytes was higher (P < 0.05) in F and H than in all vitrified/warmed oocytes, and H-VIT-Nx had the highest degeneration rate (P < 0.05). RT-PCR analysis confirmed the detection for both reference genes, and target genes BCL2 and Survivin in all samples. In contrast, BAX and p53 transcripts were consistently undetectable. No significant differences were observed in the expression of BCL2 and Survivin between groups. In conclusion, overnight holding at uncontrolled room temperature can alter oocyte characteristics and lead to variable results after vitrification. Nx demonstrated contrasting antioxidant effects depending on the vitrification timing, but it appeared to improve IVM outcomes in oocytes vitrified immediately after collection.
{"title":"Effects of holding and the addition of naloxone on vitrification of equine immature oocytes","authors":"Penelope Maria Gugole , Augusta Zannoni , Monica Forni , Eleonora Iacono , Filippo Zambelli , Barbara Merlo","doi":"10.1016/j.theriogenology.2025.02.025","DOIUrl":"10.1016/j.theriogenology.2025.02.025","url":null,"abstract":"<div><div>This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10<sup>−8</sup> M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10<sup>−8</sup> M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At survival rate determination, the degeneration rate was higher in VIT and VIT-Nx compared to F (P < 0.05). The highest maturation rate was observed in VIT-Nx. A significant reduction in ROS levels was observed in H compared to F (P < 0.05). ROS levels were similar between F and VIT, while the Nx supplementation tended to increase them (VIT-Nx vs F: P = 0.053; VIT-Nx vs VIT: P = 0.069). Conversely, in oocytes vitrified after overnight holding, vitrification induced an increase in ROS levels (H vs VIT: P < 0.05), which was not observed in H-VIT-Nx. GSH intracellular levels showed significant differences only in held oocytes, with higher GH levels in H compared to H-VIT and H-VIT-Nx (P < 0.05). All treatments induced an increase in HMMP levels compared to F (P < 0.05). In H oocytes, mitochondria were distributed throughout the entire oolemma (TOMM20) and active mitochondria (D-LAT) were detected in the outermost region. Incontrast, in H-VIT-Nx, potentially active mitochondria were spread throughout the cytoplasm. AnnexinV/PI staining revealed that the percentage of viable oocytes was higher (P < 0.05) in F and H than in all vitrified/warmed oocytes, and H-VIT-Nx had the highest degeneration rate (P < 0.05). RT-PCR analysis confirmed the detection for both reference genes, and target genes <em>BCL2</em> and <em>Survivin</em> in all samples. In contrast, <em>BAX</em> and <em>p53</em> transcripts were consistently undetectable. No significant differences were observed in the expression of <em>BCL2</em> and <em>Survivin</em> between groups. In conclusion, overnight holding at uncontrolled room temperature can alter oocyte characteristics and lead to variable results after vitrification. Nx demonstrated contrasting antioxidant effects depending on the vitrification timing, but it appeared to improve IVM outcomes in oocytes vitrified immediately after collection.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117359"},"PeriodicalIF":2.4,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143620836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-27DOI: 10.1016/j.theriogenology.2025.02.024
Janaina Menegazzo Gheller , Wilian Aparecido Leite da Silva , Mirela Brochado Souza-Cáceres , Aldair Félix da Silva , Mariane Gabriela Cesar Ribeiro Ferreira , Taynara dos Santos Santana , Angélica Camargo dos Santos , Sérgio Antonio Pereira-Junior , Érkilis Nogueira , Sérgio Amorim de Alencar , Gustavo Guerino Macedo , Marcelo Marcondes Seneda , Marcos Roberto Chiaratti , Fabiana de Andrade Melo-Sterza
While antral follicle count (AFC) has been associated with higher pregnancy rates, at present, our understanding of it as a reproductive parameter remains incomplete. This study aimed to characterize gene expression profile of oocytes from crossbred Bos taurus x Bos indicus heifers with high and low AFCs. Crossbred Nelore-Angus heifers (n = 50) with a mean (SD) age of 9.6 ± 0.55 months, a weight of 295.4 ± 32.6 kg, and a BCS of 3.44 ± 0.41 were studied in a feedlot system. The heifers received a hormonal protocol based on injectable progesterone and estradiol cypionate administered 12 days apart, and ovarian ultrasonography (US) was performed 12 days after to assess the AFC. Based on AFC, heifers were divided into low (≤14 follicles) and high (≥31 follicles) AFC, groups.Forty-five days after US, 14 heifers were slaughtered, and their ovaries were collected for morphological analysis and follicle aspiration. Cumulus-oocyte complexes (COCs) from the high and low AFC groups were graded according to their quality. Only best-quality COCs were stored for RNA-seq analysis. No differences were found in the presence or diameter of the dominant follicle and corpus luteum in the US, nor in the volume of the dominant follicle postmortem. The quantity of COCs recovered from high-AFC heifers was higher than that from low-AFC heifers (P < 0.05), and a tendency (P = 0.07) toward a higher amount of grade II COCs was observed. Thirty-two genes were differentially expressed between the groups, of which 30 were up-regulated and two down-regulated in the low AFC group. Among these, 22 % (7/32) were associated with fertility (CAB39, SLC2A6, CITED2, FDX1, HSD11B2, CD81, and PLA2G12B). Moreover, 9 and 2 exclusive genes were identified in the high and low AFC groups, respectively. Enrichment analyses showed that genes exclusive to oocytes from low-AFC heifers were associated with fundamental cellular processes, such as biosynthesis/biogenesis of ribosomes, peptides, amides, and nucleotides, and also with autophagy, mitophagy and mTOR signalling pathways.On the other hand, only one pathway was enriched in the high AFC group, but this cannot be related to the events studied No differences were observed in the ovarian structures after pre-synchronization of the estrus cycle of young Crossbred Nelore-Angus heifers. However, a tendency of a higher amount of grade II COCs was observed in heifers with high AFC than in those with low AFC. RNA sequencing results indicated that the main differences between high and low AFC heifers were not reflected in the genes directly related to fertility.
{"title":"Transcriptomic analysis of heifers according to antral follicle count","authors":"Janaina Menegazzo Gheller , Wilian Aparecido Leite da Silva , Mirela Brochado Souza-Cáceres , Aldair Félix da Silva , Mariane Gabriela Cesar Ribeiro Ferreira , Taynara dos Santos Santana , Angélica Camargo dos Santos , Sérgio Antonio Pereira-Junior , Érkilis Nogueira , Sérgio Amorim de Alencar , Gustavo Guerino Macedo , Marcelo Marcondes Seneda , Marcos Roberto Chiaratti , Fabiana de Andrade Melo-Sterza","doi":"10.1016/j.theriogenology.2025.02.024","DOIUrl":"10.1016/j.theriogenology.2025.02.024","url":null,"abstract":"<div><div>While antral follicle count (AFC) has been associated with higher pregnancy rates, at present, our understanding of it as a reproductive parameter remains incomplete. This study aimed to characterize gene expression profile of oocytes from <em>crossbred</em> Bos taurus x <em>Bos indicus</em> heifers with high and low AFCs. Crossbred Nelore-Angus heifers (n = 50) with a mean (SD) age of 9.6 ± 0.55 months, a weight of 295.4 ± 32.6 kg, and a BCS of 3.44 ± 0.41 were studied in a feedlot system. The heifers received a hormonal protocol based on injectable progesterone and estradiol cypionate administered 12 days apart, and ovarian ultrasonography (US) was performed 12 days after to assess the AFC. Based on AFC, heifers were divided into low (≤14 follicles) and high (≥31 follicles) AFC, groups.Forty-five days after US, 14 heifers were slaughtered, and their ovaries were collected for morphological analysis and follicle aspiration. Cumulus-oocyte complexes (COCs) from the high and low AFC groups were graded according to their quality. Only best-quality COCs were stored for RNA-seq analysis. No differences were found in the presence or diameter of the dominant follicle and corpus luteum in the US, nor in the volume of the dominant follicle postmortem. The quantity of COCs recovered from high-AFC heifers was higher than that from low-AFC heifers (P < 0.05), and a tendency (P = 0.07) toward a higher amount of grade II COCs was observed. Thirty-two genes were differentially expressed between the groups, of which 30 were up-regulated and two down-regulated in the low AFC group. Among these, 22 % (7/32) were associated with fertility (CAB39, SLC2A6, CITED2, FDX1, HSD11B2, CD81, and PLA2G12B). Moreover, 9 and 2 exclusive genes were identified in the high and low AFC groups, respectively. Enrichment analyses showed that genes exclusive to oocytes from low-AFC heifers were associated with fundamental cellular processes, such as biosynthesis/biogenesis of ribosomes, peptides, amides, and nucleotides, and also with autophagy, mitophagy and mTOR signalling pathways.On the other hand, only one pathway was enriched in the high AFC group, but this cannot be related to the events studied No differences were observed in the ovarian structures after pre-synchronization of the estrus cycle of young Crossbred Nelore-Angus heifers. However, a tendency of a higher amount of grade II COCs was observed in heifers with high AFC than in those with low AFC. RNA sequencing results indicated that the main differences between high and low AFC heifers were not reflected in the genes directly related to fertility.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"237 ","pages":"Pages 178-187"},"PeriodicalIF":2.4,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143520135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Our previous studies have identified phoenixin-14 (PNX-14) and its receptor GPR173 in the porcine corpus luteum (CL) during the estrous cycle and their role in the endocrine function. This study explored PNX-14's impact on luteal angiogenesis, proliferation, and apoptosis. Luteal cells were cultured with PNX-14 at doses 1–1000 nM for 24–72 h. Then, the transcript level and secretion of angiogenic factors (VEGFA, bFGF2, ANG-1) and protein expression of their receptors (VEGFR1, VEGFR2, FGFR1, FGFR2, TIE2) were analysed. Cell proliferation was assessed using the alamarBlue assay, whereas DNA fragmentation and caspase 3/7 activity through Cell Death Detection ELISA and CaspaseGlo 3/7 assay, respectively. We also examined mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclins, and apoptotic factors. Using pharmacological inhibitors of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), 5′AMP-activated protein kinase (AMPK), and silencing of GPR173 by siRNA we checked their involvement in PNX-14 action in CL. The results showed that PNX-14 increased levels of bFGF2 and ANG-1, and protein expression of VEGFR2, FGFR1, and TIE2, while it decreased FGFR2. It enhanced luteal cell proliferation and PCNA expression, with variable effects on transcript and protein levels of cyclins. Moreover, PNX-14 decreased DNA fragmentation and caspase 3/7 activity, expression of caspases 3, 8, 9, and BAX, and increased BCL2. Additionally, GPR173 receptor and ERK1/2, AKT, and AMPK are involved in PNX-14 action on luteal function. In conclusion, PNX-14 acts as a luteotropic factor in the porcine CL by promoting angiogenesis, proliferation, and protection against apoptosis.
{"title":"Effect of phoenixin-14 on angiogenesis, cell proliferation, and apoptosis in porcine corpus luteum: Role of GPR173 and ERK1/2, AKT, and AMPK signal pathway","authors":"Ewa Mlyczyńska , Karolina Pich , Joelle Dupont , Agnieszka Rak","doi":"10.1016/j.theriogenology.2025.117366","DOIUrl":"10.1016/j.theriogenology.2025.117366","url":null,"abstract":"<div><div>Our previous studies have identified phoenixin-14 (PNX-14) and its receptor GPR173 in the porcine corpus luteum (CL) during the estrous cycle and their role in the endocrine function. This study explored PNX-14's impact on luteal angiogenesis, proliferation, and apoptosis. Luteal cells were cultured with PNX-14 at doses 1–1000 nM for 24–72 h. Then, the transcript level and secretion of angiogenic factors (VEGFA, bFGF2, ANG-1) and protein expression of their receptors (VEGFR1, VEGFR2, FGFR1, FGFR2, TIE2) were analysed. Cell proliferation was assessed using the alamarBlue assay, whereas DNA fragmentation and caspase 3/7 activity through Cell Death Detection ELISA and CaspaseGlo 3/7 assay, respectively. We also examined mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclins, and apoptotic factors. Using pharmacological inhibitors of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), 5′AMP-activated protein kinase (AMPK), and silencing of GPR173 by siRNA we checked their involvement in PNX-14 action in CL. The results showed that PNX-14 increased levels of bFGF2 and ANG-1, and protein expression of VEGFR2, FGFR1, and TIE2, while it decreased FGFR2. It enhanced luteal cell proliferation and PCNA expression, with variable effects on transcript and protein levels of cyclins. Moreover, PNX-14 decreased DNA fragmentation and caspase 3/7 activity, expression of caspases 3, 8, 9, and BAX, and increased BCL2. Additionally, GPR173 receptor and ERK1/2, AKT, and AMPK are involved in PNX-14 action on luteal function. In conclusion, PNX-14 acts as a luteotropic factor in the porcine CL by promoting angiogenesis, proliferation, and protection against apoptosis.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"238 ","pages":"Article 117366"},"PeriodicalIF":2.4,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143549541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-27DOI: 10.1016/j.theriogenology.2025.117363
Josep M. Cambra , Cristina A. Martinez-Serrano , Heriberto Rodriguez-Martinez , Juan M. Vazquez , Cristina Cuello , Maria A. Gil , Emilio A. Martinez , Inmaculada Parrilla
Embryo transfer (ET) has transformed swine biotechnology, enabling genetic advancements and disease control. However, its success remains inconsistent, partly due to immune-mediated challenges at the maternal-fetal interface. This study explores the immunological environment of hemi-allogeneic pregnancies (via artificial insemination, AI) versus allogeneic pregnancies (via ET) in pigs during the critical implantation phase. Sows were categorized into groups based on pregnancy type and fetal counts, reflecting varying outcomes. Endometrial immune cell populations, including T lymphocytes, regulatory T cells (Tregs), natural killer (NK) cells, and macrophages, were analyzed using immunohistochemistry. Notably, allogeneic pregnancies with poor outcomes displayed elevated NK cell and macrophage infiltration alongside reduced Treg presence, contributing to a pro-inflammatory environment. In contrast, allogeneic pregnancies with favorable outcomes exhibited immune profiles resembling hemi-allogeneic pregnancies, suggesting enhanced maternal-fetal tolerance. These findings underscore the immunological variability driving embryo survival rates in ET pregnancies and highlight key cellular targets for improving reproductive efficiency. Further research on immunomodulatory strategies is vital to optimize ET success in swine and other species.
胚胎移植（ET）改变了猪的生物技术，使基因进步和疾病控制成为可能。然而，其成功率仍不稳定，部分原因是母胎界面存在免疫介导的挑战。本研究探讨了半异体妊娠（通过人工授精）与异体妊娠（通过 ET）在猪的关键植入阶段的免疫环境。根据妊娠类型和胎儿数将母猪分为几组，以反映不同的结果。采用免疫组化方法分析了子宫内膜免疫细胞群，包括 T 淋巴细胞、调节性 T 细胞（Tregs）、自然杀伤细胞（NK）和巨噬细胞。值得注意的是，结果不佳的异体妊娠显示了NK细胞和巨噬细胞浸润的升高，同时Treg的存在减少，从而造成了一种促炎环境。相比之下，预后良好的异体妊娠则表现出与半异体妊娠相似的免疫特征，表明母胎耐受性增强。这些发现强调了驱动 ET 妊娠胚胎存活率的免疫学变异性，并突出了提高生殖效率的关键细胞靶点。进一步研究免疫调节策略对优化猪和其他物种的 ET 成功率至关重要。
{"title":"Deciphering immune tolerance in allogeneic pig pregnancy","authors":"Josep M. Cambra , Cristina A. Martinez-Serrano , Heriberto Rodriguez-Martinez , Juan M. Vazquez , Cristina Cuello , Maria A. Gil , Emilio A. Martinez , Inmaculada Parrilla","doi":"10.1016/j.theriogenology.2025.117363","DOIUrl":"10.1016/j.theriogenology.2025.117363","url":null,"abstract":"<div><div>Embryo transfer (ET) has transformed swine biotechnology, enabling genetic advancements and disease control. However, its success remains inconsistent, partly due to immune-mediated challenges at the maternal-fetal interface. This study explores the immunological environment of hemi-allogeneic pregnancies (via artificial insemination, AI) versus allogeneic pregnancies (via ET) in pigs during the critical implantation phase. Sows were categorized into groups based on pregnancy type and fetal counts, reflecting varying outcomes. Endometrial immune cell populations, including T lymphocytes, regulatory T cells (T<sub>regs</sub>), natural killer (NK) cells, and macrophages, were analyzed using immunohistochemistry. Notably, allogeneic pregnancies with poor outcomes displayed elevated NK cell and macrophage infiltration alongside reduced T<sub>reg</sub> presence, contributing to a pro-inflammatory environment. In contrast, allogeneic pregnancies with favorable outcomes exhibited immune profiles resembling hemi-allogeneic pregnancies, suggesting enhanced maternal-fetal tolerance. These findings underscore the immunological variability driving embryo survival rates in ET pregnancies and highlight key cellular targets for improving reproductive efficiency. Further research on immunomodulatory strategies is vital to optimize ET success in swine and other species.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"238 ","pages":"Article 117363"},"PeriodicalIF":2.4,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143521109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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