Deep Intrauterine Insemination Research Articles

Inseminación artificial con semen congelado equino: reacción inflamatoria, transporte espermático y técnica de inseminación

This manuscript presents a brief historical review of investigations related to equine artificial insemination with frozen semen. The sperm-uterine interaction can be modified by the type of the insemination: concentration, total sperm numbers, motility/viability of sperm, volume, frequency and timing of AI. The frozen semen AI is widely used in the equine industry. The minimum recommended dose of spermatozoa has been established in 250 x 106 progressive motile sperm for uterine body insemination. However, the minimum dose required for maintaining the highest fertility for each stallion may differ greatly and therefore is determined by individual variation of intrinsic stallion fertility as well as with the insemination site. Deep intra-uterine insemination is commonly accepted as a routine procedure for AI with low doses. The number of AIs per cycles does not seem to be a factor affecting the final outcome of pregnancy as long as the last AI is performed within 12 h before and 12 h after the ovulation. Contrary to the common belief, the mare’s oocyte remains viable and fully capable of fertilization for 12-15 h, and the embryo loss rate after post-ovulatory insemination does not begin to increase until 12 h post-ovulation. Therefore, in a postovulatory insemination regime, examination of mares at intervals of any less than 12 h does not improve pregnancy or embryo loss rates.

Swine artificial insemination: development and biotechnology applications

Traditional artificial insemination has been highly contributed in swine global industry development. However, in order to improve the productivity and the reproductive performance of pig farms, new techniques have been performed. Among them, post cervical or intrauterine insemination, deep intrauterine insemination (DUI), intra-oviductal insemination (ΙΟΙ) by laparotomy or laparoscopy, as well as the biotechnological applications, such as sex sorted semen, frozen semen and sperm mediated genes' transfer, have been investigated concerning their efficiency in pig production development. One of the main targets of the aforementioned techniques is to benefit the potential advantages of the high genetic value boars by using the minimal number of spermatozoa needed to achieve a high fertilization rate following artificial insemination. On the other hand, biotechnology could lead in rapid transfer of high genetic potential in inaccessible areas through frozen semen. Moreover, biotechnology could support pig industry increasing the female pigs' production according to the consumers' choices and animals' welfare by sex sorted semen application, as well as producing of transgenic pigs aiming an increased disease resistance, improvement carcass composition, increased feed intake and growth rate. In vivo studies have evaluated the efficiency of the new developments in insemination technology. The results of these studies demonstrated that a satisfactory conception rate and litter size can be obtained by the insemination of low number of spermatozoa in the appropriate position of the female genital tract at the right time, close to ovulation. High farrowing rate and number of live born piglets were noticed after the post cervical insemination with low number of spermatozoa (lxlO9), compared to the traditional artificial insemination. Similar results were observed in other experiments, where 150xl06 or 3xl05 spermatozoa were inseminated using DUI or ΙΟΙtechnique, respectively. Furthermore, the improvement of the materials of the new artificial insemination procedures (catheters, extenders etc) makes the application of it simpler and easier for the workers after a few training. Although the traditional artificial insemination is an indispensable method for the commercial pig farms, the application of currently available sperm technologies is feasible to be performed in selected animals of pig farms with high quality control and management system. The aspects of the development on swine artificial insemination technology concerning the biotechnological applications of boar sperm are reported in the present study.

Pregnancy Outcome Using Highly Concentrated, Cooled Stored Stallion Semen and Different Dilution Protocols With Autologous Seminal Plasma Before Insemination

Reducing seminal plasma (SP) is essential in stallion semen preservation; however, SP has an important role in sperm fertility as well as sperm protection and transportation in the female genital tract. In the present study, semen storage at high concentrations and low volumes and the effect of SP added immediately before insemination, volume, and deposition (corpus or uterine horn) of the insemination dose were evaluated. Semen processing protocols were investigated in experiment 1: dilution to a final volume of 20 mL (control); or centrifugation and dilution of the sperm pellet to a volume of 1.5 mL containing 500 × 106 sperm. Immediately before insemination, centrifuged samples were diluted with INRA96 extender containing 0%, 5%, 20%, or 80% SP to a final volume of 20 mL. In experiment 2, concentrated semen was not further processed before insemination. Pregnancy rates were lowest when adding 20% SP to the semen extender (100%, 89%, 67%, 30%, and 67% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Embryonic growth between days 12 and 14 was highest using 0% SP (292%, 515%, 290%, 343%, and 404% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Uterine fluid accumulation did not differ among groups (P > .05). The number of progressively motile sperm per dose was similar in pregnant (median, range: 347, 217–452) and nonpregnant cycles (314, 195–369; P > .05). Deep intrauterine insemination did not improve pregnancy outcome when using 500 × 106 sperm (P > .05). Stallion semen can be stored at 5°C for 24 hours highly concentrated with low volumes of extender, yielding normal pregnancy rates. Adding SP to ejaculated sperm immediately before insemination is not necessary after 24 hours of cooled storage.

EFFICIENCY OF INTRAUTERINE INSEMINATION IN BREEDING SOWS

Improving of fertility indicators and reproductive qualities of sows during artificial insemination in farms of different specializations is very topical issue. The introduction of artificial insemination of sows on breeding farms by the spermdoses of optimum volume, by the frozen-thawed and sex sperm requires the introduction of innovative reproductive technologies. The use of economical methods of artificial insemination of sows using a minimum number of sperm in a small volume of spermdose in order to achieve high rates of fertility and prolificacy was proven in numerous experiments of the authors. Significantly reduced spermdose may be sufficient if the sperm enters deep enough into the uterus. Vitality of sperm does not depend on the size of spermdose, but the best place for sperm to survive one oviducts where they keep the fertilizing capacity from 9 to 27 hours. So deep intrauterine insemination of sows improves conditions for sperm survival The aim was to study the feasibility and justification for widespread implementation in to production on breeding farms of intrauterine insemination of sows morder to increase their fertilization and prolificacy and to save the boar sperm with the highest index of breeding values. Experiments were conducted in terms of selection and genetic center of Agrofirm "Mig-Service-Agro" in Mykolaiv region. In the experiment used 65 sows of live weight of 280-320 kg wiht 2-4 farrowing were. Sows in sexual hunting were showed once daily in the morning using a boar-prober. Artificial insemination was performed twice: the first time - in the afternoon and at 14-16 p.m. The second time – in the morning of the next day at 9-10 am. For artificial insemination of sows were ed using experimental spermodes with volume of 40 ml which contained 1.5 billion of a Active sperm. For the dilution of sperm was used Durasperm - KRUUSE (Denmark) the period of sperm perpetuation is 5-7 days. To enter the were semen used catheters Magaplus S, (Spain) for intrauterine insemination of sows. Analysis shows that the period from weaning to insemination has significant difference comparing sows of large White breed with Landrace breed (p &lt;0.001), with genotype sows F1 (p &lt;0.01) and sows of the Duroc breed (p &lt;0.05). For all selected 65 sows duration of suckling period, was estimated which averaged 32.2 days and the average time from weaning of pigs to their sexual inclination and the first intrauterine insemination 6.8 days that meets the physiological norm. After intrauterine insemination of sows of then 48 farrowed, which wich made for 73.9%. іncluding live 5 emergency farrow were obtained, representing 10.4% of all amount. Percentage of farrow is considered physiologically normal – 80%, or more of total insemined sows. The very low percentage of farrow 53.3% had of sows F1, and the highest percentage was found by sows of the Duroc breed – 85.7%. Pregnency of sows were received just 17, which made 26.1% and highest percentage – 46.7% was set by sows F1. Analysis of the pregnancy sows shows that on average it is 116.2 days was the longest – 117.1 days was set in Landrace breed sows and the short est 115.5 days in Large White breed, but the difference is not significant. 571 pigs were received, іncluding live 451 head, which is 78.9%. The largest percentage of іncluding live piglets obtained from sows F1 – 82.5%, and the lowest in Landrace breeds – 77.0% and Large White – 77.3%. Exit of all piglets per sow without emergency farrowings is 12.2, іncluding live – 9,8. The highest yield were obtained piglets from sows F1 – 13.1, іncluding live – 10.9, the lowest yield of sows of the Duroc breed – 10.6, іncluding live 9.1, which has significant difference compared with the control (IDPs) and other breeds. After intrauterine insemination 8 sows showed cyclic deregulation in 20-25 days, ie repeated sexual hunt took place on average 22.3 days. These sows were inseminated by not fractional way, they farrowed and an average litter just 13.1 piglets per sow, іncluding live - 11.3 was obtained. Repeating after intrauterine insemination on 45-48-49 day in the sexual hunt came about three sows for artificial insemination by not fractional method 3 farrowed and was obtained output – 13.3 piglets, іncluding live – 11.7. It should be noted that the best sow Large White breed №12 after intrauterine insemination bore 16 pigs, іncluding live 11, sow of Landrace breed №1556 – 18 pigs, іncluding live 12, Duroc №5888 – 13 pigs, іncluding live 11, sow F1 №167 – 20 pigs, іncluding live 14 pigs. Breeding requires more careful handling with major sows taking into account their breeding value and cost, that’s why we believe that there is no need to risk causing injury genitals with intrauterine insemination if a sufficient number of spermdoses of boars-sires exist.

Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows

We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5×108 frozen-thawed spermatozoa by DIUI at 34h after the hCG injection. At 4h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10×108 frozen-thawed spermatozoa at 36h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen.

64 COULD FROZEN–THAWED BOAR-SEMEN FERTILITY BE INCREASED WITH SEMINAL PLASMA ADDITION?

Post-thawed addition of seminal plasma (SP) in equine cryopreserved semen increased the integrity of plasma and acrosomal membranes (Andrade et al. 2011 Reprod. Dom. Anim. 46, 682–686) and these possibly affect sperm lifespan, improving fertility (Garcia et al. 2010 Anim. Reprod. Sci. 119, 160–165). This study was conducted to verify the pregnancy (PR) and fertility rate (FR) of addition of homologous SP in thawed boar semen. First, SP collections were made with polled sperm rich-fraction. Semen was centrifuged (500 × g for 10 min) and supernatant was removed, centrifuged one more time (2500 × g for 30 min), vacuum filtered through membranes (0.22 μm), and stored at –80°C for future use. Samples collected to frozen were pooled and divided in 2 aliquots, control (cryopreserved with SP; CON) and cryopreserved without SP (WSP; SP was removed and discarded after centrifugation – 500 × g for 10 min). Samples were extended in freezing extender (Botupharma®) to a final concentration of 300 × 106 spermatozoa per milliliter, packaged in 0.5-mL straws, and frozen in an automatic system (TK Tecnologia em Congelação®) using a rate of –0.5°C min–1 until 5°C, –20°C min–1 until –120°C, and then immersed in LN (–196°C). Ten straws, from each treatment, were thawed in water bath (37°C/30 s) and extended in Beltsville thawing solution to obtain 1.5 × 109 sperm in 40 mL. The other 10 straws of WSP were thawed and extended in Beltsville thawing solution plus 10% (v:v) of SP to originate treatment TSP (thawed added of seminal plasma). Thirty-three (11 per treatment) gilts had synchronized ovulation with altrenogest (25 mg/5 mL, Regumate®) administration per 18 days. Following day after withdrawal the altrenogest was administered with an intramuscular injection of 600 IU of eCG (Novormon®) and 2.5 mg of pLH (Lutropin®-V) after 72 h; a single deep intrauterine insemination was made 36 h after. Five days after, females were slaughtered and embryos were collected (by oviducts flushed) and evaluated by esteromicroscopia. Fertility rate and PR were analysed by SAS program (SAS Institute Inc., 2010, Cary, NC, USA). Fertility rate was analysed by Mixed models, and treatment effects were analysed by orthogonal contrasts (C1: effect without SP = CW v. NC; C2: effect of post-thawed addition of SP = CP v. CW), and PR was evaluated by binary distribution with PROC GLIMMIX test. Fertility rate was not affected (P &gt; 0.05) by cryopreservation of boar semen in presence or absence of SP nor by its addition in Beltsville thawing solution (10.58 ± 3.92, 9.57 ± 4.92, 21.29 ± 7.37 for CON, WSP, and TSP, respectively). Treatments did not influence (P &gt; 0.05) PR (50.00, 36.36, 72.73 for CON, WSP, and TSP, respectively). Thus, neither SP addition in thawed boar semen nor cryopreservation with or without SP can be beneficial to PR and FR, in our experimental conditions. However, a numeric large difference was observed. Therefore, these results lead us to believe that SP have a potential to increase the fertility and pregnancy rate, and that can be verified in further studies, with more repetitions.Research was supported by Agroceres Multimix, Botupharma and FAPESP process 2013/08070-8 and 2011/23484-8.

Migração espermática em suínos após inseminação artificial intrauterina profunda

A inseminação artificial intrauterina profunda (IIP) é de grande importância para a indústria suinícola, em função do maior número de doses produzidas por reprodutores de alto mérito genético e da possibilidade da utilização de biotecnologias, como sêmen sexado e/ou congelado. Entretanto, necessita-se compreender com maior propriedade os mecanismos pelos quais os espermatozoides colonizam as tubas uterinas. Assim sendo, pretende-se com o presente experimento avaliar a existência ou não de migração intraperitoneal de espermatozoides inseminados profundamente em um dos cornos uterinos, mediante a obtenção de oócitos fertilizados no corno contralateral à inseminação e seccionado na base, na junção com o corpo do útero. Quatorze fêmeas pluríparas foram divididas em dois grupos experimentais, sendo que em um deles as fêmeas foram submetidas à secção da base de um dos cornos uterinos (Grupo Operado, n = 7), enquanto as do Grupo Controle (n = 7) não foram submetidas a nenhuma intervenção cirúrgica. Ambos os grupos foram submetidos à IIP, sendo as fêmeas abatidas 5±1,2 dias após a última inseminação. Os sistemas genitais das fêmeas foram coletados, dissecados e o número de corpos lúteos contados em ambos os ovários. A recuperação dos embriões foi feita por meio de lavagem das tubas e cornos uterinos com solução de PBS (Phosphate Buffered Saline), após o que se avaliou os fluidos coletados em lupa para a identificação de embriões. Em ambos os grupos experimentais, foram encontrados embriões nos segmentos do sistema genital de ambos os lados. Apenas uma fêmea apresentou embriões nos segmentos em somente um dos lados no grupo operado. Diante dos resultados aqui observados, concluiu-se que a migração espermática no suíno pode ocorrer tanto por via retrógrada pelo útero quanto por migração intraperitoneal. Estes achados certamente contribuirão para aumentar a eficiência da técnica de IIP, sendo de grande valia para o aprimoramento da indústria suinícola.

Effect of artificial insemination protocol and dose of frozen/thawed stallion semen on pregnancy results in mares.

Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen-thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi-flexible Minitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p=0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p=0.0123).

Sperm Migration in Pigs after Deep Intrauterine and Intraperitoneal Insemination

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.

Infiltration of Local Immune Cells in the Sow Reproductive Tracts after Intra-Uterine and Deep Intra-Uterine Insemination with a Reduced Number of Spermatozoa is Less than Conventional Artificial Insemination

The present study investigated the infiltration of leukocyte subpopulations in the utero-tubal junction (UTJ) and each part of the oviducts at about 24 hr after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) compared to conventional artificial insemination (CAI) in sows. Fifteen crossbred Landrace x Yorkshire multiparous sows were used (CAI, n=5; IUI, n=5; DIUI, n=5). The sperm dose contained 3,000 × 10(6) (100 ml), 1,000 × 10(6) (50 ml) and 150 × 10(6) (5 ml) motile spermatozoa for CAI, IUI and DIUI, respectively. The sows were inseminated with extended fresh semen at 6 to 8 hr prior to the expected time of ovulation. At 25.2 ± 1.6 hr after insemination, the oviducts and the UTJ were collected. The tissue samples of UTJ, caudal isthmus, cranial isthmus and ampulla were transversely cut to a thickness of 5 µm and stained with H&E. The total numbers of lymphocytes, neutrophils, macrophages, eosinophils and plasma cells were determined under light microscope. It was found that the numbers of lymphocytes, eosinophils and macrophages after CAI, IUI and DIUI were not significantly different (P>0.1) in both epithelial and sub-epithelial connective tissue layer of the UTJ, caudal isthmus, cranial isthmus and ampulla. Intra-epithelial neutrophils in the UTJ were higher than cranial isthmus (P<0.05) and ampulla (P<0.05). In the UTJ, the intra-epithelial neutrophil in the CAI group was higher than DIUI group (P<0.01). Plasma cells in sub-epithelial layer of the endosalpinx in the CAI group were higher than DIUI group (P<0.05) and tended to be higher than the IUI group (P=0.08). In conclusion, compared to CAI, IUI and DIUI do not influence the infiltration of lymphocytes, macrophages and eosinophils in the UTJ and the oviduct prior to fertilization. But a lower number of neutrophils in the intra-epithelial layer of the UTJ and plasma cells in the sub-epithelial layers of the oviduct was observed in the DIUI group compared to CAI.

Fertility in Single‐ovulating and Superovulated Dairy Heifers after Insemination with Low Dose Sex‐sorted Sperm

The present study was performed to test fertility in single-ovulating and superovulated dairy heifers after insemination with low dose sex-sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10(6) ) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10(7) non-sorted sperm) and that of deep insemination with low dose non-sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non-sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra-uterine insemination with low dose sorted sperm was similar to that of non-sorted sperm, which was either also deposited at a low dose deep intra-uterine or into the uterine body. Synchronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non-sorted sperm.

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.

Fertilization Rate and Number of Embryos on Day 2 after Intrauterine and Deep Intrauterine Insemination Using Frozen-Thawed Boar Semen in Multiparous Sows

The present study determines fertilization rate and number of embryos on Day 2 after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) using frozen-thawed (FT) boar semen in multiparous sows. Twelve crossbred Landrace × Yorkshire multiparous sows were included. The sows were inseminated at 24 h after oestrus detection and reinseminated every 12 h until ovulation took place. The inseminations were conducted using IUI with 2 × 109 FT sperm per dose (n = 6) and DIUI with 1 × 109 FT sperm per dose (n = 6). The sows were slaughtered at 45.1 ± 7.2 h after ovulation. Embryos and unfertilized oocytes were flushed from the oviducts. IUI yielded a better fertilization rate than DIUI (66.0% versus 31.0%, P < .001). The number of embryos was 13.5 ± 2.7 and 6.6 ± 3.2 embryos/sow in IUI and DIUI groups, respectively (P = .08). The proportion of sows having unilateral fertilization was higher in the DIUI (3/5) than the IUI group (1/6). In conclusion, IUI with at least 2 × 109 total number of FT boar spermatozoa is recommended.

Expression of Progesterone Receptor in the Utero-tubal Junction After Intra-uterine and Deep Intra-uterine Insemination in Sows

The aim of this study was to investigate the expression of progesterone receptor (PR) in the utero-tubal junction (UTJ) of sows at 24 h after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) compared with conventional artificial insemination (AI) in pigs. Fifteen multiparous sows were used: AI (n = 5), IUI (n = 5) and DIUI (n = 5). The sows were inseminated with a single dose of diluted semen during the second oestrus after weaning at 6-8 h prior to ovulation (AI: 3000 × 10(6) spermatozoa, IUI: 1000 × 10(6) spermatozoa and DIUI: 150 × 10(6) spermatozoa). The UTJ was collected and subject to immunohistochemical staining using avidin-biotin immunoperoxidase technique with mouse monoclonal antibody to PR. In the oviductal part of the UTJ, the intensity of PR in the tunica muscularis and the proportion of PR-positive cells in the surface epithelium after DIUI were lower than AI (p < 0.05). The intensity and the proportion of PR-positive cells between AI and IUI in all compartments of the UTJ did not differ significantly (p > 0.05). When comparing between tissue compartments, prominent staining was observed in the muscular layer of the UTJ. It could be concluded that the expression of PR in the UTJ prior to fertilization after DIUI with a reduced number of spermatozoa was lower than that after AI. This might influence sperm transportation and the fertilization process.

Number of Spermatozoa in the Crypts of the Sperm Reservoir at About 24 h After a Low-Dose Intrauterine and Deep Intrauterine Insemination in Sows

The aim of this study was to investigate the number of spermatozoa in the crypts of the utero-tubal junction (UTJ) and the oviduct of sows approximately 24 h after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) and compared with that of conventional artificial insemination (AI). Fifteen crossbred Landrace x Yorkshire (LY) multiparous sows were used in the experiment. Transrectal ultrasonography was performed every 4 h to examine the time of ovulation in relation to oestrous behaviour. The sows were inseminated with a single dose of diluted fresh semen by the AI (n = 5), IUI (n = 5) and DIUI (n = 5) at approximately 6-8 h prior to the expected time of ovulation, during the second oestrus after weaning. The sperm dose contained 3000 x 10(6) spermatozoa in 100 ml for AI, 1,000 x 10(6) spermatozoa in 50 ml for IUI and 150 x 10(6) spermatozoa in 5 ml for DIUI. The sows were anaesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the proximal part of the uterine horns (1 cm) on each side of the reproductive tracts were collected. The section was divided into four parts, i.e. UTJ, caudal isthmus, cranial isthmus and ampulla. The spermatozoa in the lumen in each part were flushed several times with phosphate buffer solution. After flushing, the UTJ and all parts of the oviducts were immersed in a 10% neutral buffered formalin solution. The UTJ and each part of the oviducts were cut into four equal parts and embedded in a paraffin block. The tissue sections were transversely sectioned to a thickness of 5 mum. Every fifth serial section was mounted and stained with haematoxylin and eosin. The total number of spermatozoa from 32 sections in each parts of the tissue (16 sections from the left side and 16 sections from the right side) was determined under light microscope. The results reveal that most of the spermatozoa in the histological section were located in groups in the epithelial crypts. The means of the total number of spermatozoa in the sperm reservoir (UTJ and caudal isthmus) were 2296, 729 and 22 cells in AI, IUI and DIUI groups, respectively (p < 0.01). The spermatozoa were found on both sides of the sperm reservoir in all sows in the AI and the IUI groups. For the DIUI group, spermatozoa were not found on any side of the sperm reservoir in three out of five sows, found in unilateral side of the sperm reservoir in one sow and found in both sides of the sperm reservoir in one sow. No spermatozoa were found in the cranial isthmus, while only one spermatozoon was found in the ampulla part of a sow in the IUI group. In conclusion, DIUI resulted in a significantly lower number of spermatozoa in the sperm reservoir approximately 24 h after insemination compared with AI and IUI. Spermatozoa could be obtained from both sides of the sperm reservoir after AI and IUI but in one out of five sows inseminated by DIUI.

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2–3% in the freezing media, cooling rates of −30 to −50 °C/min, and thawing rates of 1200–1800 °C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1 × 10 9 spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

New developments in low-dose insemination technology

New nonsurgical procedures for inseminating swine with a low number of spermatozoa have been developed and/or evaluated over the last few years. These procedures allow the deposition of the insemination dose into the uterine body (post-cervical insemination) or directly into the uterine horn (deep intrauterine insemination). With the use of the post-cervical insemination, a threefold reduction in the number of fresh sperm has been successfully used to achieve pregnancy. Using deep intrauterine insemination (DUI), up to a 20-fold reduction in the number of fresh spermatozoa or a sixfold reduction in the number of frozen/thawed spermatozoa can be achieved, with reproductive performance very similar to that obtained after standard AI. Complementing these nonsurgical insemination techniques, a new procedure for depositing spermatozoa into the oviduct by laparoscopy has been recently described. This laparoscopic technique has proven to be applicable to diluted and sex-sorted spermatozoa. The development of new insemination procedures will help achieve more efficient application of currently available sperm technologies. Using appropriate insemination procedures, it is now feasible to achieve high fertility rates with cooled, frozen–thawed, or sex-sorted semen.

Low‐Dose Insemination in Pigs: Problems and Possibilities

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.

Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig

The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6-8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 x 10(6) motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 x 10(6) motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 x 10(6) motile spermatozoa. The sows were anesthetized 61.1 +/- 12 h after insemination (48-72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 +/- 2.6 per sow and the embryos numbering 11.4 +/- 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48-72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts.

Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig

Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig