Andréia Maria Silva

Over the last two decades, reproductive research conducted primarily in Neotropical wildlife species from the Brazilian Caatinga has evolved from descriptive studies of reproductive biology into an integrated framework for biodiversity conservation. The trajectory described herein illustrates how basic reproductive physiology, when systematically linked to assisted reproductive technologies (ARTs), can generate practical conservation tools. Initial investigations focused on reproductive anatomy, endocrinology, spermatogenesis, follicular dynamics, and reproductive behavior. These studies provided the foundation for the development of species-specific methods for semen collection, sperm evaluation, estrous monitoring, hormonal manipulation, cryopreservation, in vitro culture, and xenotransplantation. Subsequent advances expanded conservation efforts from gamete preservation to gonadal tissue biobanking, ultimately supporting the establishment of integrated genetic resource conservation strategies. Using species from the Caatinga as experimental models, including collared peccaries, red-rumped agoutis, Spix’s yellow-toothed cavies, six-banded armadillos, greater rheas, and other taxa, this research program generated knowledge applicable to a broad range of Neotropical wild species. Beyond summarizing scientific achievements, this article presents lessons learned during twenty years of research and discusses future opportunities involving advanced biobanking and the use of emerging assisted reproduction technologies. The experience demonstrates that long-term, species-centered programs can serve as effective pathways for transforming biological knowledge into conservation solutions.

Bees are essential pollinators with species differing morphologically and physiologically. Understanding the variations in reproductive parameters between phenotypes is crucial. This study compares the sperm characteristics of Italian (Apis mellifera ligustica) and Africanized drones (Apis mellifera L.) raised in the Caatinga biome. Nine sexually mature Italian drones and sixteen Africanized drones from different colonies were used. Semen was collected using the endophallus eversion technique and diluted in saline solution (1:20). The parameters analyzed included: motility (optical microscopy), sperm viability (Hoechst 33342; propidium iodide), functional integrity of the plasma membrane (hypo-osmotic test), morphology and morphometry (Rose Bengal), and scanning electron microscopy. The results were expressed as mean ± standard error. Statistical analyses included the Shapiro-Wilk test to the normality of residuals and the Bartlett test to verify homoscedasticity. Comparisons between groups were performed using the Mann-Whitney and Student's t-tests (P < 0.05). Both phenotypes presented 90% sperm motility with viability of 82.4 ± 2.5% for Italians and 81.1 ± 2.4% for Africanized ones; the functional integrity of the plasma membrane was 93.4 ± 1.8% and 91.6 ± 1.5%, respectively. Regarding morphology, the percentage of normal sperm was 10.89 ± 1.66% for Italian and 12.06 ± 1.01% for Africanized, with the curled tail being the most predominant feature of sperm morphology. No statistically significant differences (P > 0.05) were observed for the above-mentioned parameters. Sperm head morphometry was significantly larger (P < 0.05) in Italian (10.04 ± 0.03 µm) compared to Africanized (9.33 ± 0.04 µm). Scanning electron microscopy analysis revealed no ultrastructural differences between phenotypes. In conclusion, there is a high degree of similarity in sperm parameters of both phenotypes under the same environmental conditions, indicating the feasibility of applying similar reproductive strategies.

The cryopreservation of male gonadal tissue is critical to conserve genetic material and use it later via assisted reproduction. This study aimed to evaluate cryopreservation methods (slow freezing, SF; solid surface vitrification, SSV) as well as the optimal concentrations of intracellular cryoprotectants during the SSV of testicular tissue from prepubertal collared peccaries. Five pairs of testes were dissected on different days into small fragments (3 mm³) and allocated to a non-cryopreserved, a control group or one of three treatment groups: SF; SSV 3 M (1.5 M dimethyl sulfoxide [DMSO] plus 1.5 M ethylene glycol [EG]); or SSV 6 M (3 M DMSO plus 3 M EG). After one week of storage in liquid nitrogen, tissue samples were warmed and evaluated in terms of histology, viability, proliferative capacity potential, and DNA integrity. The scores for histological integrity and cellular damage for SF (2.08 ± 0.05 and 2.33 ± 0.07, respectively) were similar to the results found in SSV 6 M (1.93 ± 0.04 and 2.30 ± 0.07; p > 0.05). However, these scores were better when compared to SSV 3 M (1.87 ± 0.05 and 2.08 ± 0.06; p < 0.05). The percentage of cellular viability was around 57% after all preservation treatments (p > 0.05), which was lower than in the control group (88.8 ± 1.9%; p < 0.05). The SSV 6 M treatment was better than the other treatments regarding the proliferative capacity potential of spermatogonia cells (3.52 ± 0.03) (p < 0.05), although it was lower than in the control group (4.00 ± 0.12) (p < 0.05). Additionally, SSV 6 M led to the same DNA integrity (97.0 ± 0.7%) as in the control group (99.4 ± 0.3%). These collective findings suggest that the combination of SSV with 6 M cryoprotectants is the most efficient for the cryopreservation of testes from prepubertal collared peccaries.

O cervo rusa (Rusa timorensis) é uma espécie tropical originária da Ásia, atualmente distribuída no Brasil, Malásia, Tailândia, Nova Caledônia, Austrália e Ilhas Maurício. Estudos sobre reprodução, incluindo análises ultramicroscópicas, são essenciais para a compreensão da morfologia espermática específica da espécie e para abordar possíveis problemas reprodutivos que afetam os esforços de conservação. Dois cervos rusa machos adultos foram sedados com um dardo contendo cetamina (2 mg/kg) e xilazina (1 mg/kg). A coleta de sêmen foi realizada por eletroejaculação e uma alíquota de 60 μL de cada amostra foi fixada em solução de Karnovsky modificada. As amostras foram enviadas para avaliação em microscópio eletrônico de varredura e microscópio eletrônico de transmissão, onde foi possível observar a superfície celular e suas partes, estruturas internas, e o comprimento e largura da cabeça, peça intermediária e cauda, medindo 8,68, 5,55, 13,89 e 36,69 µm, respectivamente. As estruturas internas observadas incluíam a bainha mitocondrial, fibras densas externas, axonema, núcleo, acrossomo e membrana plasmática. Tais achados evidenciam que os espermatozoides do cervo rusa compartilham características estruturais com os de outros mamíferos, como suínos, humanos e porquinhos-da-índia, além de exibirem particularidades morfométricas que podem ser úteis para aprimorar estratégias de conservação e reprodução assistida. Esses dados não apenas destacam a relevância de preservar a espécie, mas também oferecem bases para pesquisas futuras, visando à otimização de técnicas reprodutivas e à manutenção da diversidade genética em cervídeos.

We verified the possibility of cooling peccary semen for 4, 24, and 48 h before cryopreservation, using different dilution media (TRIS + egg yolk (20%) and PRIMXcell Ultra). Ten ejaculates were divided equally into six aliquots and then diluted. Two aliquots were stored in a biological incubator (4 h), and the remaining aliquots were stored in a commercial container, the Botutainer^® (24 and 48 h), both at 5 °C. The samples were cryopreserved and then evaluated for kinetic parameters, functionality, integrity, mitochondrial activity, morphology, and sperm binding capacity. After thawing, samples diluted in TRIS showed total motility of 43.4 ± 6.8%, 48.4 ± 6.2%, and 38.6 ± 5.0% after cooling for 4, 24, and 48 h before cryopreservation, respectively. Such results are significantly greater than those achieved with the use of PRIMXcell diluent for 4 (8.3 ± 2.8%), 24 (4.7 ± 1.4%), and 48 h (4.8 ± 2.9%) storage (p < 0.05). Furthermore, TRIS provided better preservation of sperm membrane integrity when samples were cooled for 24 h (44.5 ± 4.7%) before cryopreservation compared to those samples diluted in PRIMXcell Ultra stored for 24 (25.7 ± 4.0%) and 48 h (25.2 ± 4.0%) before freezing (p < 0.05). In summary, we suggest TRIS diluent + egg yolk (20%) as an effective option to allow semen to cool for 24 or 48 h in a transport container before cryopreservation.

The effects of antibiotics on sperm longevity in collared peccary (Pecari tajacu) fresh diluted semen was evaluated. Semen samples from six adult males were collected by electroejaculation and diluted in Tris-citrate-fructose alone (control) and plus streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml). Membrane integrity and functionality, mitochondrial activity and sperm morphology were assessed subjectively. Sperm motility and other kinetic parameters were objectively assessed using CASA (computer-assisted semen analysis). The semen diluted according to the treatments were submitted to the thermoresistance test, incubated at 37 ° C, and the sperm parameters analyzed at 0, 30, 60, 120 and 180 min. The average values of the treatments were compared with each other and between the times. There were no differences (P > 0.05) between treatments until the end of the test. Control and streptomycin-penicillin samples maintained sperm function for up to 180 min (with total motility of 24.3 ± 7.1% and 28 ± 8.7%, respectively). Gentamicin aliquots retained most parameters until the end of the incubation, except for membrane integrity and mitochondrial activity that declined (P < 0.05) at 180 min (53.1 ± 7.1% and 50.7 ± 6.2%, respectively) compared to 0 min (80.5 ± 4.7% and 86.3 ± 3.4%, respectively). In conclusion, a multiparametric thermoresistance test proved that Tris-based extenders used for collared peccary semen can be effectively supplemented by streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml), especially during 180-min incubation at 37 °C.

The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 10⁶) compared to the epididymis (201.0 ± 77.4 × 10⁶). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.

1. The objectives of this study were to establish the use of the fluorophores Hoechst 33342 and propidium iodide for the evaluation of sperm plasma membrane integrity and to identify an adequate hypoosmotic solution for the evaluation of sperm membrane functionality in quails. 2. Sperm samples were collected from the vas deferens of nine quails. After initial evaluation, the samples were subjected to a flash-frozen assay. Three treatments with the following proportions of fresh sperm and sperm subjected to flash freezing were prepared as follows: 100:0 (T100), 50:50 (T50), and 0:100 (T0). The hypoosmotic swelling test used distilled water (0 mOsm/l) and fructose solutions (50, 100, and 200 mOsm/l). 3. Immediately after recovery, the samples showed 75.6 ± 5.0% motility with vigour of 3.7 ± 0.3 and 96.1 ± 0.5% of the sperm appeared normal. The membrane integrity test showed 62.2 ± 5.2% intact sperm at T100, 29.0 ± 4.1% at T50 and 0.1 ± 0.1% at T0. Moreover, a greater number of reactive sperm (74.7 ± 6.7%) were observed when incubated in distilled water (0 mOsm/l) in comparison to other solutions (P<0.05). 4. The association of fluorescent probes composed of Hoechst 33342 and PI provided an efficient assessment of the integrity of the plasmatic membrane of quail spermatozoa. However, the sgtudy identified that HOST has little predictive value regarding sperm membrane functionality in this species.

We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen–thawed sperm. Semen aliquots from ten individuals were diluted in a Tris–egg yolk–glycerol extender alone or with 0.5% Equex STM^® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris–yolk–glycerol extender optimized the freeze–thaw recovery of peccary semen.