Cryptorchid Pigs Research Articles

Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs

Introduction: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. Methods: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. Results: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. Conclusion: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.

The Use of Abdominal Ultrasound to Improve the Cryptorchidectomy of Pigs.

Simple SummaryCryptorchidism is the failure of one or both the testes to descend in the scrotum. In pigs, the incidence of cryptorchidism varies from 4% to 12%, and the most frequent localisation of retained testes is the abdominal region. In other species, transabdominal ultrasound is the most reliable diagnostic tool to localize the retained testis. This study aimed to evaluate the value of ultrasound in correctly identifying the location of retained testes, to improve the cryptorchidectomy in pigs. The ultrasound examination reduced the duration of anaesthesia, surgery, and postoperative pain. Indeed, the technique used is safe, effective, and rapid. To locate the testis, it should recognise the echo structure of the testicle and the mediastinum is well always recognisable for its hyperechogenicity. The routine use of the ultrasound examination is considered a useful alternative before the cryptorchidectomy in the pig because it allows the exact location of the retained testicle, ensuring reduced manipulation of the viscera.This study aimed to describe a technique to locate retained testes in pigs by means of ultrasound examination and thereafter proceed with cryptorchidectomy. Fifty-two monolateral cryptorchid pigs were enrolled. After anaesthesia, 42 pigs (Group U) underwent ultrasound examination before cryptorchidectomy, and 10 pigs (Group C) were used as control group without ultrasonography. The total duration of anaesthesia, ultrasound examination, and surgery was evaluated. In 10 pigs of Group U and 10 pigs of Group C, the heart rate (HR), respiratory rate (RR), and body temperature (T) were monitored to assess intraoperative pain response. An operator used the Piglet Grimace Scale (PGS) to assess postoperative pain. In Group U, the total time required for anaesthesia and surgery was less than Group C. No intra- or postoperative complications were reported in both groups. For HR, RR, and T, no statistically significant differences were observed. During the postoperative pain assessment, the highest pain scores were recorded at T6 (6 h after surgery). Ultrasound examination was found to be a valid technique for locating the retained testis in the abdomen in cryptorchid pigs and to reduce the duration of the surgery.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

BackgroundCryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry.ResultsA PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age.ConclusionsThe present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

Is the Piglet Grimace Scale (PGS) a Useful Welfare Indicator to Assess Pain after Cryptorchidectomy in Growing Pigs?

Simple SummaryOn farms, pigs undergo husbandry procedures that can be painful. However, pain assessment in pig farming faces practical limitations, pertaining to the lack of reliable and feasible tools. This work aimed to investigate whether the Piglet Grimace Scale (PGS), a facial-expression-based pain coding system, could be usefully applied to assess pain associated with on-farm surgery in growing pigs. Ten pigs affected by cryptorchidism (a congenital anomaly which requires surgical intervention) were assessed before and 6 h after surgery. The authors found that the PGS score increased after surgery, and therefore, that the PGS is a potentially effective method to assess pain associated with surgery in growing pigs.Pig cryptorchidism is a congenital anomaly, which requires surgical intervention. Pain assessment in pig farming has some limitations and consumers are increasingly concerned about suffering linked to on-farm procedures. The PGS seems to be a promising tool for pain assessment in piglets, although it has not been investigated whether this tool is applicable to growing pigs. This study was designed to determine whether the PGS could be useful to assess pain in growing pigs undergoing on-farm cryptorchidectomy. Ten mixed-breed cryptorchid pigs were pre-medicated with azaperone and ketamine. Anesthesia was induced and maintained using IV sodium based. Pigs were filmed pre- and 6 h post-surgery to evaluate their behavior (scan sampling every minute). Besides, 36 pictures of the face expressions (18 pre- and 18 6 h post-surgery) were scored with the PGS by three treatment-blind observers. The pre-surgery pig’s activity ratio was 59%, while it was 2% 6 h post-surgery. While the PGS inter-observer reliability was excellent (Interclass Correlation Coefficient value of 0.87), the PGS score increased significantly in pigs 6 h post-surgery (Paired sample t-test, p = 0.02). The PGS proved to be a potentially effective method to assess pain associated with cryptorchidectomy. However, further validation studies are required to validate this tool for other potentially painful procedures.

Expression patterns of male germ cell markers in cryptorchid pig testes

Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

Alteration of androgen receptor expression, apoptosis and cell proliferation in cryptorchid suckling, nursery and growing-finishing pigs

Cryptorchidism, a condition of one or two undescended testicles, is a common congenital disease in pigs, causing loss in the pig industry. One of the major factors affecting testicular descent is the androgen receptor (AR), which binds to androgen and then regulates the expression of androgen-responsive genes in the inguinoscrotal phase of testicular descent. AR expression has been reported to regulate apoptosis in testicular stem cells. The present study aimed to immunohistochemically examine AR and Ki-67 protein expression and apoptosis detection in unilateral undescended testicles (UDT) and descended testicles in cryptorchid pigs (DT) of suckling (aged 1–2 weeks), nursery (aged 6 weeks) and growing-finishing pigs (aged 12, 15 and 20 weeks) and in normal testicles (NT) at 1–2 and 12 weeks of age. At 1–2 weeks, decreased expression of AR was observed in UDT and DT compared with NT and was lower than that at 6–20 weeks. The expression of Ki-67, a marker of cell proliferation, in UDT and DT at 12 weeks was lower than that in NT at the same age. In addition, Ki-67 expression in UDT at 6 and 12 weeks was lower than that in UDT at 1–2 and 15–20 weeks. More testicular apoptosis was revealed in UDT at 1–2 weeks than in DT and NT at the same age. At 15–20 weeks, more apoptosis was detected in UDT than in DT. Positive correlation of AR expression in DT at 6 and 12 weeks was also noted, in addition to the association of the expression of AR and Ki-67 in NT at 12 weeks. Taken together, this study unveiled the low expression of AR and high apoptosis detection in UDT, whereas low expression of AR and low apoptosis detection were noted in DT in suckling piglets. Diminished cell proliferation was shown in UDT at 6–12 weeks, whereas high apoptosis was observed in UDT at 15–20 weeks. High expression of AR was shown only in nursery pigs. Distinct expression of AR in DT and NT at 1–2 and 12 weeks indicated that both conditions were not interchangeable.

Pig surgery: cryptorchidectomy using an inguinal approach

The objective of the study was to determine whether a less-invasive surgical technique used in cryptorchid horses, dogs and cats can be used successfully to remove retained testicles in pigs...

Suppression of boar taint in cryptorchid pigs using a vaccine against the gonadotropin-releasing hormone

Thirteen unilaterally cryptorchid Large White pigs, which had been immunized at 4 and 8 weeks of age and a third time at 64 ± 4 kg body weight against the gonadotropin releasing hormone with the vaccine Improvac®, were slaughtered at the age of 170 ± 9 days at a body weight of 102 ± 12 kg. Twelve pigs tested negative in the olfactory test of the salivary gland; their descended testicles were small and their fat androstenone concentration was low compared to normally developed boars of a previous experiment which had been vaccinated twice with Improvac® according the manufacturer's recommendation. One cryptorchid boar, which tested positive in the olfactory test and whose testicular weight and fat androstenone concentration corresponded to values of unvaccinated boars of the same age, obviously had not responded to the vaccination. It is an open question if the vaccination protocol for normal boars is sufficient to prevent boar taint in the majority of cryptorchid pigs, too.

Effect of pig sex on consumer ratings for pork chops and bacon

The study was conducted to determine whether consumer acceptability of fresh and cured pork differed among gilt, intersex, cryptorchid, and intact male market pigs. Samples of chops and bacon from 12 animals of each sex were tested using 9-point hedonic scales for sensory characteristics. Androst-16-ene steroid levels, blood levels of testosterone and estrogen were determined, as was ‘taint’ by a fry test of fat. A significant sex effect for tenderness ( P < 0·0092) and juiciness ( P < 0·0095) resulted from chops of intact males being liked least. Low consumer ratings were usually texture related. Cryptorchid pigs varied most in eating characteristics being the only sex to exhibit significant differences for liking of aroma and flavour of chops. The few significant differences obtained for bacon were not related to those obtained for chops. Fry test and steroid levels were not related to liking with the exception of testosterone which reduced mean liking.

Identification and characterization of 19-nortestosterone in urine of meat-producing animals

To monitor the illegal use of 19-nortestosterone as an anabolizing agent in meat-production, the Belgian Institute of Veterinary Expertise applies a strategy of urine control by radioimmunoassay, positive samples being confirmed by thin-layer chromatography. We have evaluated this control strategy, using gas chromatography—mass spectrometry to confirm the presence of 19-nortestosterone, or its metabolite oestrane-diol, in positive samples from radioimmunoassay. Our results show that the effective way of proceeding remains reliable in cattle, for mature and immature males as well as non-pregnant females, and in pigs, for pregnant and non-pregnant sows. The possible presence of endogenous 19-nortestosterone in cattle, in pregnant cows urine, and in pigs, in boars and in cryptorchid pigs, impedes the control of the use of 19-nortestosterone on these samples. False-positive (not confirmed by gas chromatography—mass spectrometry) results were produced by radioimmunoassay in the urine of castrated pigs and sheep.

Investigations of arterio-venous anastomoses in the spermatic cords and blood supply, oxygen consumption and testosterone production of scrotal and abdominal testes in the pig.

This study has investigated blood flow from the testicular artery to the pampiniform plexus in the spermatic cord of pigs. Testosterone levels, oxygen tension and the degree of acidity were measured in arterial and venous blood vessels of scrotally and abdominally located testes. Haemoglobin oxygen saturation was derived from the oxygen dissociation curve. Blood flow to abdominal and scrotal testes and epididymides was measured using the radioactive microsphere technique. Average blood flow to the scrotal testes and epididymides was 21 and 8 ml/min, respectively, in normal pigs. In unilaterally cryptorchid pigs average blood flow to the scrotal testis and epididymis was 23 and 6 ml/min, respectively, and to the abdominal testis and epididymis 4.2 and 1 ml/min. In pigs with bilateral scrotal testes oxygen consumption was 16 mumol O2/min/100 g. In unilaterally cryptorchid pigs oxygen consumption by the scrotal testis was 18 mumol O2/min/100 g, compared with 10 mumol O2/min/100 g by cryptorchid testes. From the percentage oxygen saturation in the various blood vessels it was calculated that 29-42% of testicular arterial blood was flowing through arteriovenous anastomoses between the testicular artery and the pampiniform plexus in the spermatic cord, thus bypassing the capillary net of the abdominal testes of unilaterally and bilaterally cryptorchid pigs. These results were supported by the testosterone measurements. In the spermatic cord of scrotal testes no blood bypassed the capillary net of the testes.

The effect of transection of the main vascular and nervous supply of the testis on the development of spermatogenic epithelium in the pig

The treatment of cryptorchidism in boys by the orchiopexy procedure is variably successful due to several technical problems. In order to study some of these problems a series of experiments in the male pig were undertaken. Experimental simulations of the orchiopexy procedure in the inguinal canal in male pigs with normally descended testes gave rise to damage of the spermatogenic epithelium. Cooling experiments of abdominal testes in adult, naturally cryptorchid pigs indicate that the arrest of spermatogenesis in abdominal testes is not due to an inborn defect but is caused by maintenance of the testes at the abdominal temperature. Evaluation of the data of the interventions with the testicular artery, vein, and nerve, either separately or in combination in the abdomen, showed that there was no effect on the spermatogenic epithelium in any one of the five experimental groups. These experiments confirm that in cryptorchid boys transection of the testicular artery and vein in the abdomen may be indicated if a tension-free fixation of the testis in the scrotum is not achievable, provided that alternate routes are able to take over the main blood supply to the testis.

The prevention of orchiopexy-induced testicular lesions in the pig.

From previous experiments it was concluded that failure of spermatogenic cells to differentiate after orchiopexy seems to be due to surgical trauma. Furthermore it appeared that in normally descended testes of naturally unilaterally cryptorchid pigs subjected to orchiopexy, the spermatogenic epithelium was poorly developed as compared with that of scrotal testes of unilaterally cryptorchid pigs that had not undergone the surgical procedure. Even simple inguinal surgery in unilaterally naturally cryptorchid piglets may already give rise to severe histological lesions in the spermatogenic epithelium of the normally descended contralateral testes at adulthood. On the other hand extirpation of the retained testis by laparotomy does not affect the spermatogenic epithelium in the contralateral scrotal testis. From these experiments it may be concluded that with regard to the prevention of sterility in unilaterally cryptorchid pigs it may be preferable to extirpate the retained testis rather than to perform an orchiopexy.

The Origin of Orchiopexy-Induced Testicular Lesions in the Pig

Cooling experiments of abdominal testes in adult, naturally cryptorchid pigs indicate that spermatogenic arrest in abdominal testes is not due to an inborn defect, but is caused solely by maintenance of the testis at abdominal temperature. It is postulated that failure of spermatogenic cells to differentiate after orchiopexy results from surgical trauma. Evaluation of the orchiopexy procedure revealed that simple manipulation of a normally descended testis may give rise to damage to the spermatogenic epithelium. Furthermore, it appeared that, in normally descended testes of naturally unilaterally cryptorchid pigs subjected to orchiopexy, the spermatogenic epithelium was poorly developed as compared with that of scrotal testes of unilaterally cryptorchid pigs that had not undergone a surgical procedure.

Induction of Spermatogenesis in the Naturally Cryptorchid Pig

Abdominal testes of adult, naturally unilaterally cryptorchid boars were subjected to continuous artificial cooling for 5, 15, 25, or 45 days. This treatment initiated development of the spermatogenic epithelium. After a cooling period of 45 days there was complete differentiation in many seminiferous tubules. The results indicate that the spermatogenic arrest in abdominally located testes is not due to an inborn defect in the testis, but is caused by the maintenance of the testis at the abdominal temperature.

Early deviations of testicular Leydig cells in the naturally unilateral cryptorchid pig.

In this study, histomorphometric, histologic and histochemical aspects of the Leydig cell development of the abdominal and scrotal testis of the naturally unilateral cryptorchid perinatal pig were examined. In the pig, Leydig cells differentiate from about 2.5 weeks before to 2.5 weeks after birth, followed by regression up to 7 weeks. Within 2 weeks after birth, initial deviations were recognized in the intertubular Leydig cells of the abdominal testis. When compared with their homologs in the scrotal testis, they revealed reduced presence of enzyme histochemical activity of 3n-HSD and 17(3-HSD, reduced number of fuchsinophilic granules, increased number of sudanophilic droplets and cellular vacuoles and changes in cellular location of 1 7j3-HSD. At about 4 weeks after birth, cell volume of both peritubular and intertubular Leydig cells was slightly reduced, but marked histomorphometrical deviations did not develop until 10 weeks after birth. Most data from the scrotal testis of the unilaterally cryptorchid pig were within the range for normal pigs. When the abdominal testis is compared with the scrotal testis, it is concluded that in the former, the postnatal part of the Leydig cell development is accomplished to a lesser degree and that the subsequent regression of the Leydig cell is accelerated. It is suggested that after birth, the temperature difference between scrotal and abdominal testes is a major factor in the development of differences between their respective Leydig cells.

Sertoli cell ultrostructure. I. A comparative study in immature, pubescent, adult and cryptorchid pigs

Sertoli cell ultrostructure. I. A comparative study in immature, pubescent, adult and cryptorchid pigs

Histomorphometric aspects of testicular morphogenesis in the naturally unilateral cryptorchid pig.

The abdominal and scrotal testes of naturally unilateral cryptorchid pigs, ranging in age from 12#{189} weeks postcoitum up to sexual maturity were investigated by histomorphometry in an attempt to determine the time of onset and subsequent development of testicular aberrations, characteristic of the cryptorchid condition. Both the abdominal and scrotal testes were compared with testes of a group of normal pigs. Testicular development in both the abdominal and scrotal testis of the cryptorchid pig progresses normally until birth. During the first month postpartum aberrations in the abdominal testis become evident. The marked perinatal development of the Leydig cells as well as the consequently occurring increase in testis weight is less pronounced. The doubling rate of the germ cells is diminished after birth. From the second month postpartum onwards the diameter of the seminiferous tubules decreases in comparison to that of the companion scrotal and to the normal testis; the pubertal increase in diameter is delayed and reduced, while the increase in length is almost ceased. Although the total number of germ cells per testis increases during the total period covered by this investigation, spermatocytes are only occasionally present during prepuberty. The companion scrotal testis becomes hypertrophic after the first month postpartum. At first this is mainly due to a more pronounced development of the Leydig cells, but at puberty all testicular components contribute equally to the hypertrophy.

"Slaughter testicle" in the cryptorchid pig.

"Slaughter testicle" in the cryptorchid pig.

A HISTOLOGICAL AND CHEMICAL STUDY OF THE FATTY MATTER OF NORMAL AND CRYPTORCHID TESTES

1. About 19 per cent. of the dry weight of the normal pig testicle is fatty matter. Histologically this fat occurs largely in the cells of the seminal tubules, and especially in the Sertoli cells. 2. During spermatogenesis the fat of the Sertoli cell passes inward for the nutrition of the spermatids and spermatozoa. During this passage its character is altered from a neutral fat to a lipoid. 3. About 30 per cent. of the dry weight of the cryptorchid pig testicle is fatty material. Histologically this fat lies within the seminal tubules, partially filling the Sertoli cells. The spermatogenic cells have completely disappeared. 4. We conclude that the presence of such an excessive amount of fat in the cryptorchid testicle is due to the absence of the spermatogenic cells which normally utilize during their development the fat furnished by the Sertoli cells.