Comparison of different intracellular cryoprotectants on the solid surface vitrification of red-rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.     

哺乳动物的卵巢冷冻保存

经冷冻保存后的卵巢或组织可以保存大量的原始卵泡,并且结构完整,具有活力及发育能力,且已直接用于临床获得妊娠。因此,卵巢冷冻近年来成了生殖生物学、生殖医学的研究热点。本文就卵巢冷冻的历史、基本技术原理、方案选择与评价及其影响因素作一综述。     

Vitrification of ovarian tissue of Brazilian North‐eastern donkeys (Equus asinus) using different cryoprotectants

The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). Ten pairs of ovaries from Brazilian north‐eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid‐surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p < 0.05). When comparing treatments, the use of DMSO 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (72.3 ± 18.0%; p < 0.05). When vitrified using the DMSO + EG combination, a higher percentage (62.5 ± 29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p < 0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3 M + EG 3 M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field fertility

Semen banking is critical to preserving rare and autochthonous breeds. However, protocols can change with time, leaving heterogeneous semen batches. The objective of this study was to assess differences in sperm quality and field fertility. We report differences between batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were analysed by CASA and flow cytometry. The 85‐days non‐return rates from AI records were used to assess the fertility of 23,853 AI. BIOXCell showed higher quality post‐thawing. Differences increased after a 5‐hr incubation at 37°C, and Biociphos yielded doses with lower resilience. Field fertility did not differ between extenders (Biociphos: 57.4% ± 1.2; BIOXCell: 56.6% ± 3.0), possibly because of AI protocols compensating for differences in quality. However, this needs to be confirmed by controlled intervention studies. In conclusion, batches frozen with Biociphos may require specific strategies for compensating for the lower sperm quality. Regular surveys and evaluation of cryobank procedures may be useful to characterizing stored batches and defining strategies to guaranteeing success in their future use.     

Ovarian remnant syndrome: revascularization of free-floating ovarian tissue in the feline abdominal cavity

Nine, healthy, intact female domestic shorthair cats were ovariohysterectomized. At the time of surgery and following removal, the major portion of one ovary was loosely sutured to the mesentery and replaced in the abdominal cavity. Six months later, an abdominal laparotomy was performed in order to retrieve the ovarian remnants. Histopathological examination of the remnants showed viable tissue and evidence of ovarian follicles or corpora lutea in eight of nine (88.9%) cats. The ninth ovarian remnant was atrophied and fibrotic. Measurement of serum estradiol and progesterone, vaginal cytology, and stimulation of estrus and ovulation with a protocol using pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were unreliable indicators of ovarian activity in this study. Revitalization of an ovarian remnant was shown to occur in the absence of surgical implantation.     

First successful frozen semen of the maned wolf (Chrysocyon brachyurus)

This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio^® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs.     

Relevance of ovarian follicular development to the seasonal impairment of fertility in weaned sows

A field study was conducted to estimate seasonal differences in follicular development in weaned sows and to evaluate the implication of these differences on seasonal infertility. A total of 110 sows were selected at weaning during winter–spring (WS, n = 58) and summer–autumn (SA, n = 52). Ovaries were scanned once daily from weaning to the onset of oestrus and twice daily from then until ovulation. Six sows during WS were removed from study for not showing growing follicles at weaning. Oestrus was evaluated twice daily from day 1 after weaning to day 14 post-weaning. One of 52 (1.9%) sows in WS and 9/52 (17.3%) in SA showed no signs of oestrus within 14 days of weaning (P < 0.05). The diameters of the follicles at weaning, at the onset of oestrus and just before ovulation were smaller (P < 0.01) in SA sows than in WS sows. There were fewer follicles in SA sows than in WS sows just before ovulation (P < 0.05). Fifty of 51 (98.0%) sows in WS and 31/43 (72.1%) sows in SA experienced a weaning-to-oestrus interval (WOI) of 3–6 days (P < 0.05). Fifty-one of 52 (98.1%) sows in WS and 43/52 (82.7%) sows in SA were inseminated; the percentage of pregnant sows that failed to farrow was lower in WS (1/51, 2.0%) than in SA (5/43, 11.6%; P < 0.05). The percentage of farrowed sows was greater in WS (46/51, 90.2%) than in SA (32/43, 74.4%; P < 0.05). Sows in WS had on average 1.5 more piglets than sows in SA (P < 0.05). Sows with a WOI of 3–6 days had lower rates of pregnancy losses (P < 0.05) and higher farrowing percentages (P < 0.01) than those with a WOI > 6 days, irrespective of season.     

Intra‐operative imaging of surgical margins of canine soft tissue sarcoma using optical coherence tomography

Optical coherence tomography (OCT) is a rapid non‐invasive imaging technique that has shown high sensitivity for intra‐operative surgical margin assessment in human breast cancer clinical trials. This promising technology has not been evaluated in veterinary medicine. The objective of this study was to correlate normal and abnormal histological features with OCT images for surgical margins from excised canine soft tissue sarcoma (STS) and to establish image evaluation criteria for identifying positive surgical margins. Fourteen client‐owned dogs underwent surgical resection of a STS and OCT imaging of 2 to 4 areas of interest on the resected specimen were performed. Following imaging these areas were marked with surgical ink and trimmed for histopathology evaluation. Results showed that different tissue types had distinct characteristic appearances on OCT imaging. Adipose tissue exhibited a relatively low scattering and a honey‐comb texture pattern. Skeletal muscle and sarcoma tissue were both dense and highly scattering. While sarcoma tissue was highly scattering, it did not have organized recognizable structure in contrast to muscle which showed clear fibre alignment patterns. In this investigation, we showed different tissue types had different and characteristic scattering and image texture appearances on OCT, which closely correlate with low‐power histology images. Given the differentiation between tissue types the results support that OCT could be used to identify positive surgical margins immediately following resection of STS. Further research is needed to assess the diagnostic accuracy of this method for surgical margin assessment.     

OPS法玻璃化冷冻山羊卵母细胞的研究

采用两种不同的冷冻保护液VS Ⅰ(EG DMSO)和VSⅡ(PROH DMSO),用OPS法对不同期的卵母细胞进行冷冻.结果表明,两种冷冻液的冷冻效果差异不显著(P＞0.05),但从数据看,VS Ⅰ要优于VSⅡ;而不同期的卵母细胞对于冷冻后的发育能力有明显的影响,无论是VS Ⅰ还是VSⅡ,GV期和培养10h卵母细胞的形态正常率、成熟率、受精率差异不显著(P＞0.05),但GV期和培养10 h卵母细胞受精率均低于ⅣM期卵母细胞,它们之间差异显著(P＜0.05).     

Morphological analysis of the reproductive activity of Curimatella lepidura (Characiformes: Curimatidae)

Curimatella lepidura, commonly known as the manjuba, belongs to the Curimatidae family. To assess the reproductive activity of this species, fish were collected from three sections of the São Francisco River: section 1 = the Três Marias reservoir (TMR), section 2 = the SFR immediately downstream of the TMR, and section 3 = the SFR 54 km downstream from the TMR after the confluence of the SFR with the Abaeté River. Fish were collected bimonthly from January to December 2012. From this, the gonadosomatic index (GSI), Fulton condition factor (K), gonadal maturation stages in females and males, and diameters of vitellogenic follicles were determined. That is, this study employed histological and histometrical techniques to study the ovaries and testes of collected fish. The Fulton condition factor was statistically higher in section 1 than in the other sections, indicating that C. lepidura presents better health conditions in this section. Fish in the maturation/mature gonadal stage were collected in the November/December and January/February bimesters, coinciding with high temperatures, a long photoperiod, and abundant rainfall in this region. The mean vitellogenic follicle diameter was statistically lowest for sections 2 and 3, with a better impact on reproduction than in section 1. Overall, the results show that C. lepidura has reproductive success in lentic environments, such as in section 1, the TMR.     

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A‐F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at ?196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.     

Effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa

The effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris–egg yolk–glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50‐ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post‐freeze‐thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris–egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm.     

Treatment with a High Dose Combination of PMSG/hCG Preparation of Mares Clinically Diagnosed with Ovarian Quiescence during the Breeding Season (Investigation from 1975 to 2000)

A total of 88 thoroughbred mares were diagnosed with clinical ovarian quiescence and subjected to four treatment regimens. Using PMSG, hCG or combinations of both. A high dose combination of 5,000IU PMSG with 5,000IU hCG showed significantly higher rates of marked estrus and ovulation induction (P<0.01) as well as conception rates (P<0.05). In the present study, the administration of a high-dose combination of PMSG with hCG was shown to be an effective treatment of ovarian quiescence in light mares.     

Histochemistry of the zona pellucida of the ovary of a species with natural polyovulation: Lagostomus maximus (Rodentia,Hystricomorpha, Chinchillidae)

This study reports the histochemistry and the distribution of glycoconjugates (GCs) in the zona pellucida (ZP) of preantral, secondary, tertiary, polyovulatory and atretic follicles of ovaries from non‐pregnant (NPr) and pregnant (Pr) females of Lagostomus maximus. GCs were studied using histochemical and lectin histochemical methods. The viscacha ZP was positive to all the histochemical techniques. In addition, it was observed that the intensity of staining of the ZP was constant in the different follicular stages between both female groups. The lectin histochemical study revealed that ZP was positive for certain lectins (WGA, RCA‐I and CON‐A) and that the labelling did not vary between the different follicular stages, but between the two groups of females. By using both histochemical techniques, it was established that the GCs present in the ZP label the complexity of the area. These results allow us to increase our knowledge on the biology of the viscacha's ovary, particularly contributing to the study of polyovulation.     

Effects of cooling rates on the quality of Prochilodus lineatus (Valenciennes, 1836) sperm

This study aims to investigate the effect of different cooling rates on the semen cryopreservation of curimba (Prochilodus lineatus). Nineteen ejaculates were obtained from adults males and cryopreserved at 15°C/min (CR15), 30°C/min (CR30) (controlled temperature inside and outside straw, speed was stable during freezing) and direct freezing in liquid nitrogen vapour (~35.6°C/min) (CRNV). The straws were thawed and seminal parameters evaluated. DNA fragmentation through the comet assay was assessed. A fresh sperm sample was not frozen and used for analyses. Data were submitted to an analysis of variance (ANOVA), and means were compared by Scott–Knott test (p < 0.05) using the R Software. Mean motility percentage was 100%, and motility duration was 39.5 ± 5.7 s for the fresh sperm (subjective analysis); 58.9 ± 8.0% and 24.5 ± 5.7 s for CR15; 64.8 ± 4.8% and 26.5 ± 7.1 s for CR30; and 50.1 ± 16% and 25.7 ± 4.7 s for CRNV, respectively. Motility percentages were higher and equal between CR15 and CR30 compared to CRNV (p < 0.05). Some sperm motion kinetics, namely average path velocity (VAP) and straight line velocity (VAS), were higher for CR30 (p < 0.05), while curvilinear velocity (VCL) and velocity progression (PRO) were lower for CRNV (p < 0.05). Straightness (STR) and wobble (WOB) were the same among treatments (p > 0.05). Sperm morphology results indicated higher means for total morphological sperm alterations in CRNV. All cooling rates caused sperm DNA fragmentation, although CR30 provided a less harmful effect. This is the first report for cryopreserved P. lineatus sperm preserved under different controlled cooling rates. The cooling rate of 30°C/min is indicated for the cryopreservation of this fish sperm as it led to the lowest detrimental spermatozoa effects.     

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis)

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1—Control fresh preantral follicles; Group 2—Vitrification treatment (Vitrification solution 1 (VS1) –TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3—vitrification treatment +5 μM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (−196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 μM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.