Abstract Background Efficient recovery of oocytes from sows may facilitate embryo production and increase the number of offspring obtained from breeding sows with desired traits. However, robust protocols for consistent oocyte retrieval in sows are still lacking. The aim of this field study was to evaluate modifications of a transvaginal OPU protocol in multiparous sows, including the use of anaesthetics and analgesics, different aspiration flow rates, and hormonal treatments. Furthermore, the effect of partial in vivo oocyte maturation prior to in vitro embryo production (IVEP) was assessed. Thirty-two hybrid sows (parity 5–7) were included in three experiments. In Experiment 1, short-term altrenogest treatment combined with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) was used to attempt synchronization of follicular development. In Experiment 2, the effects of superstimulation (1000 or 1500 IU eCG), day post-weaning, and two aspiration flow rates of 24 and 30 mL of water per minute were evaluated. In Experiment 3, OPU-derived oocytes collected 10–14 h or 34–36 h after hCG administration were subjected to IVEP to assess the effect of partial in vivo maturation. Results In Experiment 1, altrenogest-based synchronization was associated with low oocyte recovery in several animals. In Experiment 2, a mean of 30.3 ± 11.0 oocytes per OPU session was recovered from 54.8 ± 10.6 aspirated follicles, corresponding to a recovery rate of 55.3%. No significant differences were detected between the two aspiration flow rates or between superstimulation treatments. In Experiment 3, partially in vivo matured oocytes resulted in a 33% blastocyst rate, compared with 6% following full in vitro maturation. Conclusions These results demonstrate that transvaginal OPU in sedated, epidurally treated sows can yield substantial numbers of oocytes under field conditions. Partial in vivo maturation prior to IVEP was associated with improved blastocyst rates. Further controlled studies are required to optimize synchronization protocols and to improve repeatability of oocyte retrieval and IVEP.

Metabolomic analysis of boar semen associated with sexual maturation is essential for improving fertility management and breeding, with amino acids and amines playing key roles in the reproductive process. This study aimed to explore changes in amino acids and amines in boar spermatozoa and seminal plasma during puberty to sexual maturity and identify potential biomarkers of sexual maturity. Semen was collected from the same 15 Duroc boars over time at approximately 7 months (Age 1), 8.5 months (Age 2), and 10 months (Age 3). Liquid chromatography-mass spectrometry was used to analyse amino acids and amines in spermatozoa and seminal plasma separately. Multivariate analysis (PLS-DA) revealed pronounced age-dependent changes in amino acids and amines in spermatozoa between Age 1 and Age 3, and more subtle shifts in seminal plasma. Univariate analysis (Repeated measure ANOVA/Friedman) revealed that glutamate and taurine had significant pairwise differences in seminal plasma (P < 0.05). In sperm, 15 amino acids (glutamate, alanine, aspartate, choline, taurine, histidine, methionine, tryptophan, leucine, cystine, tyrosine, arginine, lysine, valine and glycine) exhibited significant pairwise differences (P < 0.05). VIP scoring (>1.5) prioritised glutamate, alanine, aspartate, and choline as key contributors to the variations and pathway analysis implicated alanine, aspartate and glutamate metabolism, and histidine metabolism linked to sexual maturity. Our study highlights metabolic changes during sexual maturation, identifying potential biomarkers for assessing reproductive maturity. These findings are initial steps toward optimising younger boars' usage in breeding, enhancing genetic gain, and reducing costs associated with their non-productive days at AI centres.

Boars undergo physiological and biochemical changes in semen composition as they grow from puberty to sexual maturity. However, comprehensive metabolomic profiles of boar semen remain uncharacterised. Understanding metabolic alterations in semen during this period is important for optimising reproductive performance in breeding programs. The aim of this study was to characterise the semen metabolome as boars mature, utilising an untargeted metabolomic approach. Semen samples were collected from 15 Duroc boars at three developmental ages: 7 months, 8.5 months, and 10 months. Sperm and seminal plasma were separated and analysed by hydrophilic interaction and reversed-phase liquid chromatography coupled with mass spectrometry to capture a wide range of metabolites. We identified a total of 4,491 features in boar semen, annotating 92 distinct metabolites. Amino acids, peptides and analogues constituted the most abundant components, followed by fatty acid esters. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) showed a clear separation between metabolomic profiles by age groups. PERMANOVA analysis of PCA scores confirmed statistically significant differences (P < 0.05) between younger (7 months) and more mature boars (8.5 months and 10 months). Pathway analysis identified porphyrin metabolism, taurine and hypotaurine metabolism, and glycerolipid metabolism as significantly enriched pathways in sperm, while glutathione and nitrogen metabolism were prominently enriched in seminal plasma. Using linear modelling, partial Spearman correlation and random forest analyses, we identified homoisovanillic acid as a key metabolite discriminating age groups in both sperm and seminal plasma. Additionally, L-glutamic acid, decanoyl-L-carnitine and N-(1,3-Thiazol-2-yl)benzenesulfonamide emerged as important sperm metabolites, while glyceric acid, myo-inositol, glycerophosphocholine, and several other compounds were identified as critical seminal plasma metabolites. This study provides a detailed characterisation of metabolic changes in Duroc boar semen during the transition from puberty to sexual maturity. Our findings enhance the understanding of reproductive development and could inform strategies to assess sexual maturity in breeding programs.

The objective of this study was to evaluate sperm quality of Duroc boars during sexual maturation and examine whether these parameters were related to metabolites in semen. Ejaculates were collected longitudinally from 28 Duroc boars at approximately 7 months of age (Age 1), 6 weeks later (Age 2) and 12 weeks later (Age 3). Motility characteristics, acrosome integrity, and viability were assessed on collection day and after five days storage at 18°C using computer-assisted sperm analysis and flow cytometry, while DNA fragmentation was measured on day five. Amino acid and amine concentrations in semen were measured by liquid chromatography-mass spectrometry and correlated with sperm quality parameters. Motility, rapid progressive motility, and acrosome intact live sperm increased significantly (adj P < 0.05) with boar maturation. Mature boars (Age 2 and Age 3) maintained superior sperm quality compared to younger boars after storage. The DNA fragmentation index was low in all samples but declined significantly from 1 % at Age 1 to 0.64 % at Age 3 (adj P < 0.05). Correlation analysis showed significant relationships between sperm quality and specific metabolites. Cystine, glutamate, aspartate, choline and taurine were inversely correlated with progressive and rapid progressive motility, while showing positive correlation with rapid non-progressive motility. These findings demonstrate that sperm quality continues improving beyond initial reproductive ability, especially between Age 1 and Age 3, with improvements in motility, viability, chromatin stability, and storage resilience. The observed relationships between metabolites and sperm quality parameters provide insights into biochemical mechanisms underlying sperm functionality during sexual maturation.

There are few existing publications that describe transvaginal ultrasound-guided Ovum Pick-Up (OPU) in sows, and the impacts of the procedure for the welfare of the animals are unknown. In this study, we evaluated the effects of OPU, performed following restraint in a claw-trimming chute, on the animal welfare and reproductive health of second parity hybrid sows. The study utilized a generalized randomized block design at a commercial sow pool. We assessed salivary cortisol levels before, during, and after the procedure to compare the physiological stress response between OPU and restraint chute procedures (control group). We found a significant increase in salivary cortisol caused by the physical restraint procedure, and that the salivary cortisol level at the end of the procedure did not differ between OPU and control groups (p = 0.51). Furthermore, we conducted a novel approach-aversion test for sows, designed to assess if a feed reward would motivate the animals to willingly participate in the OPU-procedure. The animals were trained daily to enter the chute to access a feed reward. Ten animals in each group failed to complete the training period and did not voluntarily enter the restraint chute on the experimental day. This indicates that even the short daily restraint procedure during the four-day long training period was aversive to some animals. There was no difference in aversion towards the restraint chute between OPU and control groups one day after the procedure. The reproductive performance of the animals was subsequently evaluated through oestrus synchronization and insemination of the sows after the experiment. There was no observed difference in the farrowing rate (p = 0.72) and total number of born piglets (p = 0.84) between OPU and control sows. On average, we retrieved 9.0 ± 5.9 oocytes during the OPU-sessions (N = 26). Our results show that a majority of the sows prioritize the motivation for feed over their aversion to the OPU procedure. However, the physical restraint procedure is unpleasant for the animals and elicits a temporary stress response. We suggest that transvaginal OPU may be used for the recovery of oocytes from live sows, but refinements are needed to avoid stress during the lifting procedure. Such modifications could also potentially reduce the observed inter-individual variations in oocyte recovery outcomes.

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus–oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.

Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

Hyperactive sperm motility is important for successful fertilization. In the present study, a proteome profiling approach was performed to identify the differences between Landrace boars with different levels of hyperactive sperm motility in liquid extended semen. Two contrasts were studied: (i) high versus low levels of sperm hyperactivity at semen collection day and (ii) high versus low change in levels of sperm hyperactivity after 96 h semen storage. Testicular samples were analyzed on a Q Exactive mass spectrometer and more than 6000 proteins were identified in the 13 samples. The most significant differentially expressed proteins were mediator complex subunit 28 (MED28), cell division cycle 37 like 1 (CDC37L1), ubiquitin specific peptidase 10 (USP10), zinc finger FYVE-type containing 26 (ZFYVE26), protein kinase C delta (PRKCD), actinin alpha 4 (ACTN4), N(alpha)-acetyltransferase 30 (NAA30), C1q domain-containing (LOC110258309) and uncharacterized LOC100512926. Of the differentially expressed proteins, 11 have previously been identified as differentially expressed at the corresponding mRNA transcript level using the same samples and contrasts. These include sphingosine kinase 1 isoform 2 (SPHK1), serine and arginine rich splicing factor 1 (SRSF1), and tubulin gamma-1 (TUBG1) which are involved in the acrosome reaction and sperm motility. A mass spectrometry approach was applied to investigate the protein profiles of boars with different levels of hyperactive sperm motility. This study identified several proteins previously shown to be involved in sperm motility and quality, but also proteins with no known function for sperm motility. Candidates that are differentially expressed on both mRNA and protein levels are especially relevant as biological markers of semen quality.

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 'progressively motile sperm to oocyte' ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen-thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10-12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds.

It has been debated whether intensive selection for growth and carcass yield in pig breeding programmes can affect the size of internal organs, and thereby reduce the animal’s ability to handle stress and increase the risk of sudden deaths. To explore the respiratory and circulatory system in pigs, a deep learning based computational pipeline was built to extract the size of lungs and hearts from CT-scan images. This pipeline was applied on CT images from 11,000 boar selection candidates acquired during the last decade. Further, heart and lung volumes were analysed genetically and correlated with production traits. Both heart and lung volumes were heritable, with h 2 estimated to 0.35 and 0.34, respectively, in Landrace, and 0.28 and 0.4 in Duroc. Both volumes were positively correlated with lean meat percentage, and lung volume was negatively genetically correlated with growth ( r g = − 0.48 ± 0.07 for Landrace and r g = − 0.44 ± 0.07 for Duroc). The main findings suggest that the current pig breeding programs could, as an indirect response to selection, affect the size of hearts- and lungs. The presented methods can be used to monitor the development of internal organs in the future.

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

AbstractDifferences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3–8 mm) was counted. Cumulus‐oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development.

Background Sperm hyperactive motility has previously been shown to influence litter size in pigs, but little is known about the underlying biological mechanisms. The aim of this study was to use RNA sequencing to investigate gene expression differences in testis tissue from Landrace and Duroc boars with high and low levels of sperm hyperactive motility. Boars with divergent phenotypes were selected based on their sperm hyperactivity values at the day of ejaculation (day 0) (contrasts (i) and (ii) for Landrace and Duroc, respectively) and on their change in hyperactivity between day 0 and after 96 h liquid storage at 18 °C (contrast (iii)). Results RNA sequencing was used to measure gene expression in testis. In Landrace boars, 3219 genes were differentially expressed for contrast (i), whereas 102 genes were differentially expressed for contrast (iii). Forty-one differentially expressed genes were identified in both contrasts, suggesting a functional role of these genes in hyperactivity regardless of storage. Zinc finger DNLZ was the most up-regulated gene in contrasts (i) and (iii), whereas the most significant differentially expressed gene for the two contrasts were ADP ribosylation factor ARFGAP1 and solute carrier SLC40A1 , respectively. For Duroc (contrast (ii)), the clustering of boars based on their gene expression data did not reflect their difference in sperm hyperactivity phenotypes. No results were therefore obtained for this breed. A case-control analysis of variants identified in the Landrace RNA sequencing data showed that SNPs in NEU3 , CHRDL2 and HMCN1 might be important for sperm hyperactivity. Conclusions Differentially expressed genes were identified in Landrace boars with high and low levels of sperm hyperactivity at the day of ejaculate collection and high and low change in hyperactivity after 96 h of sperm storage. The results point towards important candidate genes, biochemical pathways and sequence variants underlying sperm hyperactivity in pigs.

Finding effective predictors of traits related to boar fertility is essential for increasing the efficiency of artificial insemination systems in pig breeding. The objective of this study was to find associations between single-nucleotide polymorphisms (SNPs) within candidate genes and fertility in the breeds Landrace and Duroc. Animals with breeding values for total number of piglets born, were re-sequenced for exonic regions of 14 candidate genes related to male and female fertility using samples from 16 Landrace boars and 16 Duroc boars (four with high and four with low breeding value of total number of piglets born for each breed for male fertility, and the same for female fertility) to detect genetic variants. Genotyping for the detected SNPs was done in 619 Landrace boars and 513 Duroc boars. Two SNPs in BMPR1 and one SNP in COX-2 were found significantly associated with the total number of piglets born in Landrace. In Duroc, two SNPs in PLCz, one SNP in VWF and one SNP in ZP3 were found significantly associated with total number of piglets born. These SNPs explained between 0.27% and 1.18% of the genetic variance. These effects are too low for being used directly for selection purposes but can be of interest in SNP-panels used for genomic selection.

Background Sperm DNA integrity is considered essential for successful transmission of the paternal genome, fertilization and normal embryo development. DNA fragmentation index (DFI, %) has become a key parameter in the swine artificial insemination industry to assess sperm DNA integrity. Recently, in some elite Norwegian Landrace boars (boars with excellent field fertility records), a higher level of sperm DFI has been observed. In order to obtain a better understanding of this, and to study the complexity of sperm DNA integrity, liquid preserved semen samples from elite boars with contrasting DFI levels were examined for protamine deficiency, thiol profile and disulphide bonds. Additionally, the DNA methylation profiles of the samples were determined by reduced representation bisulphite sequencing (RRBS). Results In this study, different traits related to sperm DNA integrity were investigated ( n = 18 ejaculates). Upon liquid storage, the levels of total thiols and disulphide bonds decreased significantly, while the DFI and protamine deficiency level increased significantly. The RRBS results revealed similar global patterns of low methylation from semen samples with different levels of DFI (low, medium and high). Differential methylation analyses indicated that the number of differentially methylated cytosines (DMCs) increased in the low-high compared to the low-medium and the medium-high DFI groups. Annotating the DMCs with gene and CpG features revealed clear differences between DFI groups. In addition, the number of annotated transcription starting sites (TSS) and associated pathways in the low-high comparison was greater than the other two groups. Pathway analysis showed that genes (based on the closest TSS to DMCs) corresponding to low-high DFI comparison were associated with important processes such as membrane function, metabolic cascade and antioxidant defence system. Conclusion To our knowledge, this is the first study evaluating DNA methylation in boar sperm cells with different levels of DFI. The present study shows that sperm cells with varying levels of DNA fragmentation exhibit similar global methylation, but different site-specific DNA methylation signatures. Moreover, with increasing DNA fragmentation in spermatozoa, there is an increase in the number of potentially affected downstream genes and their respective regulatory pathways.

Contents The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n=693) and Norwegian Duroc (ND; n=655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p<0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p<0.0001), parity (p<0.05) and DFI (p<0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p<0.01). The DFI and the motility of the same semen samples were negatively correlated (p<0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p=0.0038 and 92.3% vs. 89.5%; p=0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.