Review: Sperm cryopreservation in wild small ruminants: morphometric, endocrine and molecular basis of cryoresistance.

Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample's origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent studies have revealed that the expression of aquaporins in the sperm cells of small wild ruminants could also be involved in the androgen-related seasonal variation of sperm cryoresistance. Along with epididymal and ejaculated spermatozoa, the cryopreservation of testicular tissue may provide a suitable source of male gametes, becoming an alternative for establishing germplasm banks when semen cannot be collected for whatever reason.

The effect of divergent selection for intramuscular fat on the domestic rabbit genome.

An experiment of divergent selection for intramuscular fat was carried out at Universitat Politècnica de València. The high response of selection in intramuscular fat content, after nine generations of selection, and a multidimensional scaling analysis showed a high degree of genomic differentiation between the two divergent populations. Therefore, local genomic differences could link genomic regions, encompassing selective sweeps, to the trait used as selection criterion. In this sense, the aim of this study was to identify genomic regions related to intramuscular fat through three methods for detection of selection signatures and to generate a list of candidate genes. The methods implemented in this study were Wright's fixation index, cross population composite likelihood ratio and cross population - extended haplotype homozygosity. Genomic data came from the 9th generation of the two populations divergently selected, 237 from Low line and 240 from High line. A high single nucleotide polymorphism (SNP) density array, Affymetrix Axiom OrcunSNP Array (around 200k SNPs), was used for genotyping samples. Several genomic regions distributed along rabbit chromosomes (OCU) were identified as signatures of selection (SNPs having a value above cut-off of 1%) within each method. In contrast, 8 genomic regions, harbouring 80 SNPs (OCU1, OCU3, OCU6, OCU7, OCU16 and OCU17), were identified by at least 2 methods and none by the 3 methods. In general, our results suggest that intramuscular fat selection influenced multiple genomic regions which can be a consequence of either only selection effect or the combined effect of selection and genetic drift. In addition, 73 genes were retrieved from the 8 selection signatures. After functional and enrichment analyses, the main genes into the selection signatures linked to energy, fatty acids, carbohydrates and lipid metabolic processes were ACER2, PLIN2, DENND4C, RPS6, RRAGA (OCU1), ST8SIA6, VIM (OCU16), RORA, GANC and PLA2G4B (OCU17). This genomic scan is the first study using rabbits from a divergent selection experiment. Our results pointed out a large polygenic component of the intramuscular fat content. Besides, promising positional candidate genes would be analysed in further studies in order to bear out their contributions to this trait and their feasible implications for rabbit breeding programmes.

Consensus Statement on the Psychological Needs of Patients With Chronic Inflammatory Skin Diseases. / Documento de consenso sobre las necesidades psicológicas de los pacientes con enfermedades inflamatorias crónicas dermatológicas.

OBJECTIVE: To establish recommendations to determine, identify, and manage the psychological and emotional needs of patients with chronic inflammatory skin diseases in clinical practice. METHODS: A guided discussion was held at meeting of a nominal group of expert dermatologists and psychologists on the psychological and emotional needs of patients with chronic inflammatory skin diseases, how to manage these cases, and which referral criteria to employ. Based on the results of the discussion, and with the aid of a patient focus group and a review of the literature, a master document was drawn up with recommendations for discussion. A Delphi survey was circulated among a larger group of dermatologists and psychologists to assess the level of agreement with the recommendations. RESULTS: Ten recommendations were established and can be summarized as follows: explore the psychological sphere of the patients with open questions at the time of diagnosis and during the course of the disease; provide patients with clear explanations that address their concerns and inform them of the existence of patient associations; investigate symptoms of anxiety and depression and, if confirmed by means of a Hospital Anxiety and Depression score (HADS) of 11 or greater, consider referral to a mental-health specialist; and, during visits, create a climate of trust, empathize with patients, agree goals and treatment options with them, and motivate them to adhere to those treatments. CONCLUSIONS: These recommendations may help health care professionals address psychological and emotional aspects of their patients in daily clinical practice.

Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds.

Many hundreds of exotic species and domestic animal breeds have been lost over the course of the last few decades. In order to avoid a similar fate to other animals threatened with extinction, it is crucial to develop and apply rescue strategies to ensure their survival for the future. One option as a safeguard measure is the cryopreservation of the main source of female gametes enclosed within the ovary: the primordial follicles. So far, there are three options to cryopreserve small ovarian follicles: whole ovary, ovarian cortical tissue or isolated follicles, with the use of slow freezing or vitrification methods. After cryopreservation, the harvested material can be transplanted or cultured, with the aim to produce mature fertilizable oocytes. The objective of this review is to summarize the current status of the cryopreservation of ovarian tissue in domestic species and non-endangered wild mammals as model for threatened and endangered species and breeds, and to provide new insights into techniques that can be applied in the future.

Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure.

The feasibility of repeated collection and enzymatic isolation of large numbers of viable primordial and primary follicles from living donor cows were tested. Ovarian cortical biopsies were collected transvaginally by the Biopsy Pick-Up (BPU) device, a modification of an Ovum Pick-Up instrument. Follicles were enzymatically isolated from the retrieved cortical tissue samples, and follicle viability was determined by a live/dead fluorescent assay. Six cows were subjected to BPU once per week during 4 consecutive weeks, and in each BPU session 4 cortical tissue samples were collected per ovary. Over the 4-week trial period, a total of 1443 primordial and primary follicles were collected, 1358 (94%) of which were primordial and 85 (6%) were primary follicles. In each BPU session, an average 60.1 +/- 10.7 (mean +/- SEM) primordial and primary follicles were isolated per cow. The number of follicles varied considerably throughout the trial period and between cows. Statistical analysis of the data, however, did not support the presence of any distinct trends in the follicle yields over time or between cows. A total of 111 enzymatically isolated follicles were analyzed for viability with fluorescent probes. The vast majority of isolated follicles (92.8%) were totally viable. We conclude that the standardized BPU procedure generates sufficiently large numbers of vital primordial and primary follicles, thus validating BPU as a new tool for research into early bovine follicular development.

Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anaemia: case report.

Ovarian function after orthotopic transplantation of cryopreserved ovarian tissue has been restored in women with malignant disease. Here the techniques are adapted for a non-cancer patient. In 1999, right oophorectomy was performed in a 21 year old woman before chemotherapy, prior to bone marrow transplantation. Ovarian cortex was frozen, according to a strict protocol. After thawing, ovarian cortex was reimplanted into the ovary and in a peritoneal window close to the ovary in 2004. Four-and-a-half months after reimplantation, LH, FSH, 17beta-estradiol and progesterone levels, as well as ultrasonography, demonstrated the presence of an ovulatory cycle. After this cycle, the patient experienced two other ovulatory cycles, evidenced by FSH and 17beta-estradiol concentrations, as well as ultrasound demonstration of a follicle. Follicular development was clearly observed in both the intraovarian site (1st and 2nd cycle) and the peritoneal window (3rd cycle). Restoration of endocrine ovarian function occurred after ovarian cortical strips, biopsied and cryopreserved before chemotherapy, were reimplanted into the ovary itself and a periovarian peritoneal window.

Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.

BACKGROUND: The lifesaving treatment endured by cancer patients leads, in many women, to early menopause and subsequent infertility. In clinical situations for which chemotherapy needs to be started, ovarian tissue cryopreservation looks to be a promising option to restore fertility. In 1997, biopsy samples of ovarian cortex were taken from a woman with stage IV Hodgkin's lymphoma and cryopreserved before chemotherapy was initiated. After her cancer treatment, the patient had premature ovarian failure. METHODS: In 2003, after freeze-thawing, orthotopic autotransplantation of ovarian cortical tissue was done by laparoscopy. FINDINGS: 5 months after reimplantation, basal body temperature, menstrual cycles, vaginal ultrasonography, and hormone concentrations indicated recovery of regular ovulatory cycles. Laparoscopy at 5 months confirmed the ultrasonographic data and showed the presence of a follicle at the site of reimplantation, clearly situated outside the ovaries, both of which appeared atrophic. From 5 to 9 months, the patient had menstrual bleeding and development of a follicle or corpus luteum with every cycle. 11 months after reimplantation, human chorionic gonadotrophin concentrations and vaginal echography confirmed a viable intrauterine pregnancy, which has resulted in a livebirth. INTERPRETATION: We have described a livebirth after orthotopic autotransplantation of cryopreserved ovarian tissue. Our findings suggest that cryopreservation of ovarian tissue should be offered to all young women diagnosed with cancer.

Differential expression of two genes located on the X chromosome between male and female in vitro-produced bovine embryos at the blastocyst stage.

The potentially unbalanced expression at preimplantation developmental stages of X-linked genes might be responsible of the faster development of male than female embryos in vitro. Two genes located on the X chromosome, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), are involved in controlling the amount of oxygen radicals, and hence they might have influence in embryo development. We have quantified mRNA expression of these two genes, using in vitro fertilized-in vitro cultured male and female bovine embryos. In vitro-produced early blastocysts obtained at days 7 and 8 were collected and biopsied for gender determination, and the remaining embryos were kept in LN(2) until RNA purification. After sex determination, embryos were pooled in groups of 3 males or 3 females, and mRNA was purified. Using a semiquantitative sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we detected G6PD and HPRT mRNA expression at the early blastocyst stage in all bovine embryos analyzed. Moreover, mRNA expression of both genes studied was significantly higher in female embryos than in male embryos. The differential expression of G6PD and HPRT at these early stages confirm that sex differences are evident prior to gonadal differentiation and that preimplantation bovine embryos have sexually dimorphic gene expression at least with respect to G6PD and HPRT transcripts. These differences might be responsible of the faster development in culture of in vitro-produced male bovine that has been reported. Mol. Reprod. Dev. 55:146-151, 2000.

[Colitis induced by a food allergen. A report of 20 cases]. / Colitis inducida por alergeno alimentario. Presentación de 20 casos.

In the last years, we have appreciated a descending number of classic enteral food-protein intolerance and cases of colitis related to food proteins are increasing. In this study, 20 cases of food protein colitis (mainly related to cow's milk) are reviewed and clinical, endoscopic and histological aspects are analyzed. We found that the age at the time of the first consultation was approximately 1.5 months, with males predominating. The main symptom was bloody diarrhoea. Other causes such as perianal fissure or infectious gastroenteritis were excluded. The infants were fed breast milk and/or cow's milk formula. Some patients had low hemoglobin, but low serum albumin was infrequent. The colonoscopy showed erythema and, in some cases, loss of vascular pattern, ulcerations or bleeding. The main histological finding was an increased number of eosinophils. There was a favorable response to the dietary change. After 6 months, all of the provocations done were negative. We conclude that food-protein intolerance is one of the most probable causes of bloody stools in a young infant. Colonoscopy and a histopathological study of at least three biopsy pieces are necessary for the diagnosis. The treatment is the exclusion of the offending protein from the diet.

[Rheumatic fever and post-streptococcal arthritis. Clinical review]. / Fiebre reumática y artritis postestreptocócicas. Revisión clínica.

We report 11 patients diagnosed of Acute Rheumatic Fever (ARF), and 7 of Poststreptococcal Reactive Arthritis (PSRA): patients with group A beta hemolytic streptococcal infection and articular disease who do not fulfill the modified Jones criteria. All patients with ARF were treated with monthly prophylaxis (with penicillin G benzathine). Carditis was seen in five patients, but only one of them has developed a mitral valve insufficiency. The prophylaxis in patients with PSRA was individualized. None of these patients had clinical evidence of carditis during the acute disease, but one of them developed a mitral and aortic stenosis two years after the initial episode. The possible indication of penicillin prophylaxis in these patients with PSRA in suggested.