This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques in cooled storage of semen from livestock animals such as camelids, goats, and sheep. Facing worldwide environmental changes and a trend towards more conscious and healthy eating behaviors, the development of a stable animal breeding industry is a significant challenge for the near future. In the present review, factors influencing semen handling in camelids, goats and sheep are described and relevant methods as well as current trends to improve liquid-storage of cooled semen are discussed, including extenders, additives, cooling rates, and storage temperatures. The species-specific physiology and resulting challenges are taken into consideration. While the main problem for camelid semen processing is the relatively greater viscosity as compared with that of some other animals, the deciding factor for successful artificial insemination (AI) in goats and sheep is the site (i.e., cervical or vaginal) of semen placement in the reproductive tract. Due to the type of cervical anatomy, the penetration of the cervix when using AI instruments is rather difficult. Furthermore, the seminal plasma of small ruminants affects the interaction with milk-based extenders and egg yolk which results in species-specific regimens for cooled liquid-preservation. Comparing all three species, the greatest pregnancy rates were obtained by AI with goat semen after cooled liquid-storage for several days.

Besides classical semen parameters, semen stress tests (SSTs) are helpful tools to assess the fertilizing capacity of sperm. However, valid studies on SSTs in relation to fertility are rare because several corrections of common fertility parameters for female and male effects are required. Therefore, over a one-year period, we analyzed semen parameters of 260 ejaculates obtained from 130 Pietrain boars aged between 8 and 9 months in one AI center as well as 1521 corresponding insemination records for these ejaculates. Two consecutive ejaculates (4th and 5th) were collected from each boar and extended in DiluPorc™ BTS. In addition to routine semen evaluation, sperm motility was assessed after heat-resistance test (300 min incubation at 38 °C after seven days storage at 16 °C, HRT) and cold-resistance test (10 min incubation at 38 °C after three days storage at 6 °C, CRT). Generalized linear models (GLMs) were applied to analyze effects for the following predictors of farrowing rate (FR), number of total born (NTB) and live born (NLB) piglets: farm (P = 0.013 [FR], P = 0.001 [NTB], P = 0.023 [NLB]), parity (P = 0.679, P = 0.01, P < 0.001), weekday (P = 0.012, P = 0.08, P = 0.009) and year × season (P < 0.001, P = 0.688, P = 0.574). On boar level, GLMs revealed significant effects on FR, NTB and NLB for the predictors sow (all P < 0.001), total sperm number per dose (P = 0.007, P = 0.002, P < 0.001), total sperm motility (P = 0.002, P = 0.2, P = 0.003) and mitochondrial activity (P = 0.004, P < 0.001, P = 0.002). Moreover, FR and NTB were influenced by membrane integrity (both P < 0.001), FR and NLB by cold-resistance (P < 0.001, P = 0.043), and NTB and NLB by sperm morphology (P = 0.001, P < 0.001) and boar (both P < 0.001). NLB was additionally influenced by heat-resistance (P = 0.004) and farm (P = 0.018) and solely NTB was influenced by sperm output (P = 0.03). Boar and semen related factors explained 9% of the total variation in NTB and 7% of the total variation in NLB. Only 14.2% (n = 37) of the samples were both cold- and heat-resistant (≥65% of motile sperm). Cold- and heat-resistance were dependent factors (Chi-square, P = 0.001) and sperm motility after CRT and HRT showed a moderate positive correlation (rs = 0.40, P < 0.001, Spearman's rho). Finally, ROC curves demonstrated that neither SST can be used as a sole test for predicting the fertilizing capacity of boar ejaculates.

Liquid preservation of the cold-sensitive boar sperm at a lesser temperature than the standard 17 °C would reduce bacterial growth and minimize the use of antibiotics. There was assessment, therefore, of the capacity of individual fatty acids bound to fatty acid free BSA to improve sperm survival at 6 °C because oxidative stress and lipid degradation are prominent detrimental factors. Different effects of the fatty acids were observed. Supplementation with naturally occurring fatty acids (linolenic, linoleic, oleic, palmitoleic acid), which may become metabolically incorporated into sperm lipids, increased the number of motile and progressively motile sperm after 2 days of storage during a thermo-resistance test (5 h at 38 °C) to that of control samples preserved at 17 °C in pure Beltsville Thawing Solution. With the exception of linolenic acid, all naturally occurring fatty acids enhanced the number of sperm with active mitochondria after 3 days of storage. Palmitoleic acid was the most effective supplement with effects already present when sperm were re-warmed for 30 min after 2 and 7 days of storage. The non-endogenous, non-integrated timnodonic acid (20:5) had no effect on sperm variables. Because the application of individual fatty acids attached to BSA had differing effects in preserving boar sperm at 6 °C, the use of combinations of fatty acids could be more efficacious than with use of natural lipid supplements for low temperature preservation of sperm.

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as 'near threatened', 'vulnerable' or 'endangered' (www.cites.org), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.

ContentsThe objective of this study was to evaluate mitochondria in immature and in vitro‐matured domestic cat oocytes and to assess for the first time the effect of vitrification on mitochondrial traits. Mitochondrial distribution and aggregation were assessed using confocal microscopy after staining with the fluorescent dye—MitoTracker® Red CMXRos. Only cells at the germinal vesicle and the metaphase II stages of nuclear development, representing immature and mature oocytes, respectively, were included in our study. Our study shows that 80% of immature and 100% of mature oocytes exhibit a peripheral pattern of mitochondria distribution, indicating that, in contrast to the situation in other species, the mitochondria of cat oocytes are not dispersed throughout the cell after in vitro maturation but instead maintain a strong affinity for the oocyte periphery near the membrane. However, a loss of aggregation was observed during in vitro maturation—78% of immature oocytes showed homogeneous granulation versus only 18% of mature oocytes (p < .001). The increased intensity of MitoTracker® Red CMXRos staining after in vitro maturation (p < .05) may be tentatively attributed to an increase in mitochondrial activity but could likewise reflect a concomitant appearance of sulphhydryl groups in cytoplasm (known to be targeted by the dye). Mitochondrial distribution did not change upon vitrification; however, dye intensity decreased (p < .05) and mitochondrial aggregation was intensified in both immature and mature vitrified cat oocytes.

Antibiotics are of great importance in boar semen extenders to ensure long shelf life of spermatozoa and to reduce transmission of pathogens into the female tract. However, the use of antibiotics carries a risk of developing resistant bacterial strains in artificial insemination laboratories and their spread via artificial insemination. Development of multiresistant bacteria is a major concern if mixtures of antibiotics are used in semen extenders. Minimal contamination prevention techniques and surveillance of critical hygiene control points proved to be efficient in reducing bacterial load and preventing development of antibiotic resistance. Nevertheless, novel antimicrobial concepts are necessary for efficient bacterial control in extended boar semen with a minimum risk of evoking antibiotic resistance. Enhanced efforts have been made in recent years in the design and use of antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. The male genital tract harbors a series of endogenic substances with antimicrobial activity and additional functions relevant to the fertilization process. However, exogenic AMPs often exert dose- and time-dependent toxic effects on mammalian spermatozoa. Therefore, it is important that potential newly designed AMPs have only minor impacts on eukaryotic cells. Recently, synthetic magainin derivatives and cyclic hexapeptides were tested for their application in boar semen preservation. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and potential synergistic interaction with conventional antibiotics propel predominantly cyclic hexapeptides into highly promising, leading candidates for further development in semen preservation. The time scale for the development of resistant pathogens cannot be predicted at this moment.

Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 μL containing 20 × 106 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 106 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.

Cryobanking of gametes for later use in assisted reproduction may support the preservation of endangered species. Since the application ultrasound during the freezing process is known to alter the structure of ice crystals, we attempt here to reduce the mechanical stress during the freezing process by low-frequency sonification. We used domestic animals (bovine, porcine) as models to determine the highest tolerable ultrasound intensities for sperm. Continuous application of ultrasound during the freezing process significantly increased the survival rate of bull sperm compared to non-sonified control samples frozen under similar conditions. By applying short ultrasonic pulses at sub-zero temperatures, we were able to induce a defined crystallisation process at definite temperatures in contrast to spontaneous crystallisation random temperatures. The survival rate of bull and boar sperm was, however, not significantly dependent on the seeding temperature and could not be improved. Whether the defined ultrasound-assisted seeding is advantageous for larger cells like oocytes, remains to be investigated.

Antibiotics are of great importance for the preservation of ejaculates for livestock breading. The use of antibiotics, however, is not an appropriate compensation for a lack of hygiene standards in artificial insemination (AI) centres. Sophisticated hygiene management and the proper identification of hygienic critical control points (HCCPs) at AI centres provide the basis for counteracting the development of antibiotic resistance in contaminant bacteria and their settlement in AI centres. In recent years, efforts have been made to use antimicrobial peptides (AMPs) in the preservation of boar semen. Investigations have included the testing of synthetic magainin derivatives and cyclic hexapeptides. One prerequisite for the application of AMPs is that they have a minor impact on eukaryotic cells. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and mechanisms including synergistic interaction with conventional antibiotics have made cyclic hexapeptides highly promising candidates for potential application as peptide antibiotics for semen preservation.

The aim of this research was the selection of spermatozoa parameters related to boar fertility performance and their combination into an in vitro index. A first set (data set 1) of 36 Pietrain boars with 138 ejaculates from two seasons with 5083 single-sire inseminations from 34 farms was used to determine correlations between in vitro sperm quality parameters and fertility performance. 2970 ejaculates representing a second set (data set 2) served calculation of seasonal and age effects on semen quality. Morphological spermatozoa parameters were estimated manually with a phase contrast microscope on the day of semen collection, whereas mitochondrial activity and viability were analyzed by double-staining with rhodamine123/propidium iodide on day 2 of semen storage using flow cytometry. Sperm motility was tested on day 7 by thermoresistance (TRT) after 30min (TRT1) and 300min (TRT2) incubation at 38̊C using computer-assisted semen analysis (CASA). Correlations revealed four independent sperm quality parameters qualifying as relevant predictors of boar fertility: (i) percentage of spermatozoa with proximal cytoplasmic droplets, (ii) percentage of spermatozoa with active mitochondria, (iii) beat cross frequency of progressively motile spermatozoa in TRT1, and (iv) oscillation measure of the actual path of progressively motile spermatozoa in TRT2. There were no significant effects of sperm concentration, ejaculate volume, and total number of sperm cells per ejaculate on litter size (LS) and on pregnancy rate (PR). Our findings suggest the usefulness of sperm quality parameters based on adjusted range of methods and enable the construction of an in vitro index as a means to predicting boar fertility.